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Institute of Field and Vegetable Crops, (IFVCNS), Novi Sad, Serbia
ABSTRACT
Sunflower (Helianthus annuus L.) belongs to the Helianthus genus which is
composed of 51 species (14 annual and 37 perennial). Lowered genetic variability and
sensitivity towards large number of pathogens on cultivated sunflower, point to wild
relatives as useful breeding material. Practically from the early breeding efforts in Russia
in 1930s, first uses of wild relatives were registered, and it was intensified with
introduction of hybrids since 1970s.
Increased usage led to formation of several major collections of Helianthus species,
starting from the collection in USA. They were since enlarged with new accessions in
collecting expeditions and exchange with other gene banks. The collection at Novi Sad,
Serbia was formed in 1980 with 11 annual and 32 perennial species (over 1000
accessions) and considered as one of the largest collections worldwide. Wild species are
usually kept as seeds in cold chambers, while perennial species can also be kept as living
collections in the field.
Wild species are mostly used in sunflower breeding as a source of desirable genes
for resistance to pathogens, to find Cytoplasmic Male Sterility and Restoring fertility
genes, specific oil quality, traits for new ideotypes and herbicide resistance. The
characterization data significantly increased usability and value of collections by
facilitating their use in breeding programs thus justifying the effort of collection and
maintenance.
The divergence and heterogeneity of the genus cause considerable difficulties, such
as cross-incompatibility, embryo abortiveness, sterility and reduced fertility in
interspecific hybrids. All annual species and a large number of perennial species have
*
Corresponding author: Jovanka Atlagi, Institute of Field and Vegetable Crops, (IFVCNS), Maksima Gorkog 30,
21000 Novi Sad, Serbia, jovanka.atlagic@nsseme.com
sreten.terzic@nsseme.com
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Jovanka Atlagi and Sreten Terzi 96
been crossed with the cultivated sunflower using the conventional hybridization method.
Other methods like somatic hybridization, "in vitro" embryo culture and chromosome
doubling are less often used but can be helpful for more difficult cross combinations. An
example is the interspecific program at IFVCNS which resulted in crosses with 7 annual
species (F
1
and BC
1
F
1
- BC
4
F
1
) and 14 perennial species (F
1
- BC
2
F
1
), some of which
were included in the breeding program. The success of interspecific hybridization is
evaluated based on morphological observations, cytogenetic analysis, as well as
molecular markers.
Cytogenetic studies are used for determinations of chromosome number and
structure and analysis of meiosis-microsporogenesis and pollen viability. Such studies
made it possible to establish phylogenetic relations between wild sunflower species and
the cultivated sunflower and enabled the use of the former in sunflower breeding.
The experience gathered over such a long period of sunflower prebreeding point to
difficulties in wild sunflower collection maintenance, interspecific hybridization and
isolation of desirable genes. None the less Helianthus genus has become a model genus
for studding speciation and evolution while still being a constant source of material for
improvement of cultivated sunflower.
Key words: Cultivated sunflower, wild species, desirable traits, hybridization, cytogenetics
INTRODUCTION
Sunflower breeding has reached a plateau for a number of important agronomic traits.
The major limiting factor for further improvements of the genetic potentials for seed yield and
oil quality is the susceptibility of the sunflower to a large number of pathogens. Studies in the
field of population genetics have shown that the genetic variability of the cultivated sunflower
has been drastically narrowed.
Molecular data on the diversity of the cultivated sunflower is indicating a narrow genetic
base, and thus a limited possibility for further evolution of this economically important crop
(Rieseberg and Seiler, 1990). On the other hand, molecular studies have indicated the
presence of large variability, i.e., "primitive polymorphism", in both wild species and local
populations of sunflower.
Sunflower germplasm resources can be categorized as in situ resources (i.e., wild
population and landraces) or ex situ resources (accessions preserved in gene banks).
The term 'interspecific hybridization' implies the crossing between different species of the
same genus. This method is frequently used in plant breeding, especially when variability of a
cultivated form (primary gene pool) has been exhausted and it became necessary to search for
desirable genes in its wild relatives (secondary and tertiary gene pools). This has been the
case with the cultivated sunflower (Helianthus annuus var. macrocarpus (DC.) Ckll.) which
has been crossed with wild sunflowers (Helianthus spp.). The classical hybridization method
is typically used for that purpose, while "in vitro" embryo culture and somatic hybridization
are less frequent. Interspecific hybridization is typically used for transferring resistance to
disease agents, soil salinity and acidity, and drought as well as for finding new sources of
cytoplasmic male-sterility (CMS) and fertility restoration (Rf) genes and the development of
new sunflower ideotypes.
The development and application of sunflower cytogenetics have been associated with
utilization of the Helianthus germplasm for improvement of the cultivated sunflower genome
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Sunflower Genetic Resources 97
and the use of Helianthus genus as a model for evolutionary studies. It has progressed from
cytology, via cytotaxonomy and classical cytogenetics to molecular cytogenetic studies.
This chapter reviews systematics and taxonomy of the genus Helianthus, its genomic
structure and the usefulness of wild Helianthus species as a source of desirable genes.
Cytogenetic studies on sunflower are reviewed through the analyses of chromosome number
and appearance (karyotype), meiosis (microsporogenesis), pollen viability and CMS.
The experience gathered over a long period of sunflower pre breeding point to difficulties
in wild sunflower collection maintenance, followed by cross incompatibility, lowered fertility
or complete sterility of interspecific hybrids, difficult isolation of unwanted genes and loss of
desirable genes through backcrossing.
GENETIC RESOURCES
Sunflower Origin and Dispersion
It is believed that sunflower was the first cultivated plant in North America where Native
Americans used its seeds for food. Sunflower can adapt to different habitats, including
variable vegetation length of more than seven months in Mexico, to four months in Canada.
The first written document about sunflower in Europe comes from 1567. Initially used as a
decorative plant that impressed by its size, sunflower quickly spread across Europe, it was
recognized as an oilseed crop in Russia only. Since 1880s Russian varieties could be found on
the American market. In a short period, sunflower rose from tenth (1930) to second (1970) on
the list of oil crops, behind soybeans. The percentage of oil content from about 28% (1920)
was raised to nearly 50% (1955), but the disease problem persisted (Heiser, 1976).
The first recorded attempt of interspecific hybridization between cultivated sunflower and
wild species to increase disease resistance was made in 1915. Because of the observed
interspecies compatibility in the genus Helianthus and sources of resistance to pathogens in
wild species, the use of interspecific hybridization is considered the most effective method to
compete with the new races of pathogens and maintenance of sunflower as an important oil
crop.
Systematics and Taxonomy of the Genus Helianthus
The sunflower belongs to a large and polymorphic genus Helianthus, Asteraceae family.
In the course of the 18th and 19th centuries, a number of authors had described more than 200
species from this genus. Sunflower systematics and taxonomy have been subject to continual
changes and amendments. Heiser et al. (1969) described 66 species, 48 from North America
and 18 from South America. The former group comprises 11 annual and 37 perennial species
classified into 3 sections and 7 series. Robinson (1979) reclassified the latter group into a new
genus named Helianthopsis. The North American group of the genus Helianthus (Heiser et
al., 1969) has been reconstructed following analyses of 42 morphological traits (Schilling and
Heiser, 1981). Using the biosystematics and cluster methods, 49 species were classified into 4
sections and 6 series. Section Helianthus covers 11 annual species including the cultivated
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Jovanka Atlagi and Sreten Terzi 98
sunflower. Section Agrestis includes one annual species. Section Ciliares includes two series,
Ciliares and Pumili, each containing three perennial species from North America. Section
Corona solis, Microcephali, Atrorubens and Angustifolius (Schilling and Heiser, 1981).
The classification of Schilling and Heiser (1981) is presented herein with six
modifications as described by Seiler and Gulya (2004). First, the sectional name Atrorubens
used by Anashchenko (1974) has taxonomic priority, thus the section Divaricati E. Schilling
and Heiser is replaced by section Atrorubens Anashchenko. Second, H. exilis is recognized as
a species, as opposed to an ecotype of H. bolanderi due to information which has shown it to
be morphologically and genetically distinct. Third, the species name H. pauciflorus has
priority over H. rigidus and is treated accordingly herein. Fourth, Viguiera porteri has been
transferred to H. porteri. Fifth, H. verticillatus has been rediscovered and redescribed and is
now recognized as a species. Sixth, H. niveus ssp. canescens has been transferred to H.
petiolaris ssp. canescens. This brings the number of species to 51, with 14 annual and 37
perennial (Tables 1 and 2).
Table 1. Infrageneric classification of annual Helianthus species
Section* Species
Common
Name
Chromosome
Number (n)
Helianthus H. annuus L. Prairie 17
H. anomalus Blake Anomalous 17
H. argophyllus T.& G. Silver-leaf 17
H. bolanderi A. Gray Bolanders, Serpentine 17
H. debilis
ssp. debilis Nutt. Beach 17
ssp. cucumerifolius (T. & G.) Heiser Cucumber leaf 17
ssp. silvestris Heiser Forest 17
ssp. tardiflorus Heiser Slow-Flowering 17
ssp. vestitus (Watson) Heiser Clothed 17
H. deserticola Heiser Desert 17
H. exilis A. Gray Serpentine 17
H. neglectus Heiser Neglected 17
H. niveus
ssp. niveus (Benth.) Brandegee Snowy 17
ssp. tephrodes (Gray) Heiser Ash-Colored, Dune 17
H. paradoxus Heiser Pecos, Puzzle, Paradox 17
H. petiolaris
ssp. canescens (A. Gray) Gray 17
E. E.Schilling
ssp. fallax Heiser Deceptive 17
ssp. petiolaris Prairie 17
H. praecox
ssp. hirtus Heiser Texas 17
ssp. praecox Englm. & A.Gray Texas 17
ssp. runyonii Heiser Runyons 17
Agrestes H. agrestis Pollard Rural, Southeastern 17
Porteri H. porteri (A. Gray) J. F. Pruski Confederate Daisy, Porters 17
(*Seiler and Gulya, 2004).
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Sunflower Genetic Resources 99
Table 2. Infrageneric classification of perennial Helianthus species
Section* Series Species
Common
Name
Chromosome
Number (n)
Ciliares Ciliares H. arizonensis R. Jackson Arizona 17
H. ciliaris DC. Texas blueweed 34, 51
H. laciniatus A. Gray Alkali 17
Ciliares Pumili H. cusickii A. Gray Cusicks 17
H. gracilentus A. Gray Slender 17
H. pumilus Nutt. Dwarfish 17
Atrorubens Corona-solis H. californicus DC. California 51
H. decapetalus L. Ten-petal 17, 34
H. divaricatus L. Divergent 17
H. eggertii Small Eggerts 51
H. giganteus L. Giant 17
H. grosseserratus Martens Sawtooth 17
H. hirsutus Raf. Hairy 34
H. maximiliani Schrader Maximilians 17
H. mollis Lam. Soft, Ashy 17
H. nuttallii
ssp. nuttallii T. and G. Nuttalls 17
H. nuttallii
ssp. rydbergii (Brit.) Long Rydbergs 17
H. resinosus Small Resinous 51
H. salicifolius Dietr. Willow-leaf 17
H. schweinitzii T. and G. Schweinitzs 51
H. strumosus L. Swollen, Woodland 34, 51
H. tuberosus L. Jerusalem artichoke 51
Atrorubens Microcephali H. glaucophyllus Smith White-leaf 17
H. laevigatus T. and G. Smooth 34
H. microcephalus T. and G. Small-headed 17
H. smithii Heiser Smith 17, 34
Atrorubens Atrorubentes H. atrorubens L. Purple-disk 17
H. occidentalis
ssp. occidentalis Riddell Few-leaf, Western 17
H. occidentalis
ssp.plantagineus (T. & G.) Heiser Few-leaf, Western 17
H. pauciflorus
ssp. pauciflorus Stiff 51
H. pauciflorus ssp.
subrhomboides (Rydb.) O. Spring Stiff 51
H. silphioides Nutt. Odorous 17
Atrorubens Angustifolii H. angustifolius L. Narrow-leaf, swamp 17
H. carnosus Small Fleshy 17
H. floridanus A. Gray ex Chapman Florida 17
H. heterophyllus Nutt. Variable-leaf 17
H. longifolius Pursh Long-leaf 17
H. radula (Pursh) T. and G. Scraper, Rayless 17
H. simulans E. E. Wats. Muck, Imitative 17
H. verticillatus Small Whorled 17
(*Seiler and Gulya, 2004).
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Jovanka Atlagi and Sreten Terzi 100
The taxonomic complexity of the genus Helianthus stems from many different factors.
Natural hybridization and introgression between many of the species resulted in
morphological intergradations between otherwise distinct forms. Polyploidy in the perennial
species also contributed to the complexity of species classification in Helianthus (Jan and
Seiler, 2007).
Detailed descriptions of the species (plant habit, site, geographic distribution, period of
flowering, ploidy level, etc.) were provided by Heiser et al. (1969) and Rogers et al. (1982).
The extent of variability in the genus Helianthus has not been sufficiently studied. Heiser
et al. (1969) reported that subspecies, varieties and forms existed in some Helianthus species.
The taxonomy of Schilling and Heiser (1981) retained subspecies only for some Helianthus
species. This taxonomy is simpler to use but many researchers are baffled by the intraspecific
variability occurring in their collections.
Studies of Reiseberg and Burke (2001), Rieseberg et al. (2002, 2003) and Mandel et al.
(2013) made special contributions to the knowledge of origin and speciation of Helianthus
species. Seiler (1992) concluded that changes in plant habit and distribution of species occur
as consequences of natural adaptation and natural selection in the Helianthus genus.
Gentzbittel et al. (1992) compared their molecular classification of the genus Helianthus with
morphological taxonomy of Schilling and Heiser (1981) and concluded that they are in
agreement.
Study of Miljanovi et al. (2000), and Safti-Pankovi et al. (2005) showed a large
variability for some taxonomically stable traits in the perennial species H. giganteus and H.
maximiliani, which could even justify the recognition of new infraspecific forms.
Nonetheless, possible existence of natural hybrids and growing the accessions in a common
environment vs. their natural environment must also be considered.
Germplasm Resources Collecting
Having the wild species of Helianthus within the boundaries of the USA has facilitated
collecting sunflower germplasm. The explorations for wild sunflowers in almost 40 years
have resulted in the assemblage of a USDA-ARS collection that is the most complete
collection in the world (Marek et al., 2004). It is presently located at the National Plant
Germplasm System, Plant Introduction Station at Ames, Iowa. Currently, the wild Helianthus
collection contains 2163 accessions, about two-thirds of which are annual species. Continual
collecting of wild sunflower germplasm for preservation in gene banks is critical so that
germplasm may be readily available for research and the breeding community (Seiler and
Gulya, 2004).
Dr. Charles Heiser, Indiana University was one of the early collectors of Helianthus
germplasm (Heiser, 1947). His focus was primarily taxonomy, systematics, and evolution and
speciation of the genus. His early work formed the basis of the current knowledge and
understanding of the Helianthus genus. Later explorations were undertaken in Texas and
Oklahoma in 1963 (Seiler, 1988) where the most represented wild species was H. annuus.
During the 1970s, wild sunflowers were collected throughout the southwestern USA,
resulting with approximately 200 accessions. Most of those were annual species, represented
with wild H. annuus. An exploration for sources of rust resistance was undertaken in 1972 in
the North Central Great Plains and resulted with additional 100 accessions of mostly wild
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Sunflower Genetic Resources 101
annual species. The USDA-ARS formally established a wild Helianthus collection at
Bushland, TX, in 1976. Creation of a permanent collection greatly increased the number of
collecting expeditions for wild Helianthus. During 1976, 200 accessions were collected in
Texas and New Mexico, while in 1978, 175 accessions were added from western,
southwestern, and southeastern USA. Several short explorations were made throughout the
USA in 1979 (Seiler, 1988).
Several expeditions for collecting wild sunflower were undertaken in 1980s and one in
1991 in cooperation of USDA-ARS stations in Bushland, TX and Fargo, ND with Institute of
Field and Vegetable Crops (IFVC), Novi Sad, Yugoslavia (Table 3). In a 1984 exploration to
southern Texas, Gerald Seiler collected 32 accessions of annual H. argophyllus, H. debilis
and H. praecox.
The first collecting expedition outside of the USA was in 1994 in several provinces of
Canada. Sixty-three accessions of wild sunflower were collected. Thirty-one accessions were
annual, while 32 were perennial. Almost 40% of the accessions were H. annuus (Seiler and
Brothers, 1996).
Table 3. Wild sunflower species collecting expeditions including IFVCNS staff
(1980-1991)
Collected by Year No. of state
No. of
species
No. of
accessions
Seiler, G. (USDA-ARS)
uk, L. (YUGIFVC)
1980. 21 (USA) 37 384
Marinkovi, R. (YUGIFVC)
Miller, J. (USDA-ARS)
1984. 1 (Canada) 7 88
kori, D. (YUGIFVC)
Seiler, G. (USDA-ARS)
Rooth, (USDA-ARS)
1985. 12 (USA) 13 88
Seiler, G. (USDA-ARS)
Pomeroy, J. (USDA-ARS)
Marinkovi, R. (YUGIFVC)
1987. 6 (USA) 7 52
Dozet, B. (YUGIFVC)
Seiler, G. (USDA-ARS)
Pomeroy, (USDA-ARS)
Gavrilova, V. (SUNWIR)
1989. 6 (USA) 12 84
Dozet, B. (YUGIFVC)
Marinkovi, R. (YUGIFVC)
1990. 1(Montenegro) 1 81
Marinkovi, R. (YUGIFVC)
Seiler, G. (USDA-ARS)
Stauffer, C. (USDA-ARS)
Duhoon, S. (NBPGR)
1991. 7 (USA) 9 140
Starting from 2000, collecting expeditions were more oriented to specific
underrepresented species and filling the gaps in the current wild species collection. An
exploration to southwestern USA in 2000 was undertaken for annual species H. anomalus and
H. deserticola. Annual serpentine sunflower, H. exilis, was collected in California in 2002. In
September 2003, an exploration was undertaken to California to collect the endemic perennial
species H. californicus, while perennial species H. eggertii, H. schweinitizii, H. verticillatus
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Jovanka Atlagi and Sreten Terzi 102
and annual H. porteri were collected in 2003 in the southeastern USA (Seiler and Gulya,
2004).
The collection efforts of the USDA-ARS were supported by US, European and
International organizations involved with plant genetic resources. The obtained support also
provided resources for maintenance so that the germplasm can be distributed. These
accessions have become the basis of wild species research programs in Argentina, Russia,
India, China, Mexico and many European countries. Besides USDA collection at Ames, IA,
notable collections exist at the N.I. Vavilov Research Institute of Plant Industry, St.
Petersburg, Russia, (Gavrilova and Anisimova, 2003) Institute of Field and Vegetable Crops,
Novi Sad, Serbia, (Cuk and Seiler, 1985), Dobroudja Agricultural Institute (DAI) at General
Toshevo, Bulgaria, (Christov et al., 2001) and INRA, Montpellier, France (Serieys, 1992).
The Collection of Wild Species in Novi Sad
The collection of wild sunflower species in Novi Sad was made through 7 collecting
expeditions in the period from 1980-1991., when 917 accessions were collected. The trips
varied in the number of collected species (1-37) and accessions (52-384) (Table 3).
Of the 49 sunflower species in the genus Helianthus, the collection included 43 species.
Section Helianthus was complete, and 6 species were missing from the group of perennials.
Regrettably, 14 species have been lost in the previous period, 4 annuals and 10 perennials.
The reasons for this were in the first place different conditions for growing between the
collection and the original natural habitat (winterkill, long growing season), as well as low
self-fertility and poor viability of seed of the wild species. At present, the collection
comprises 21 perennial and 7 annual species. The total number of accessions in the collection
is 447. Reserve seeds of the accessions of annual species range from several scores to several
thousands (Table 4).
Table 4. The IFVCNS collection of annual wild sunflower species
Species
No. of accessions
No. of seeds
in gene bank date base
with seed
reserves
H.annuus 108 70 10-2539
H.petiolaris 33 25 80-9130
H.neglectus 4 4 1564-4838
H.debilis 21 13 33-5490
H.praecox 15 14 335-10710
H.argophyllus 7 7 1616-10396
H.niveus 4 3 259-5910
The situation is similar with the perennial species, where the discrepancy in the number
of accessions registered in the gene bank and the number of available reserve seed lots
indicates that a certain number of accessions have been lost. From the initial 41 accessions of
H. tuberosus, only 16 remained. In the case of H. grosserratus, 9 accessions remained from
the original 29. The number of perennial accessions maintained in the field exceeds for most
part the number of accessions having seeds in storage (Table 5). It should be mentioned that
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Sunflower Genetic Resources 103
the number of reserve seeds of the perennials varies from one seed to several hundred,
exceptionally several thousands. Compared with the reserve seeds of the annuals, the number
of reserve seeds of the perennials is considerably smaller.
Detailed information about the collected accessions including passport data can be
accessed online on the IFVCNS site: http://www.nsseme.com/about/inc/oilcrops/wild.php
Table 5. The IFVCNS collection of perennial wild sunflower species
Species
No. of accessions
No. of seeds
No. of
accessions in
the field
In gene bank
data base
With seed
reserves
H.tuberosus 41 16 1-650 112
H.rigidus (H.pauciflorus) 13 12 1-400 11
H.mollis 7 5 3-1162 6
H.maximiliani 36 32 1-8630 60
H.divaricatus 10 10 8-680 8
H.decapetalus 8 7 17-622 7
H.grosseserratus 29 9 2-335 31
H.nuttallii 23 22 1-399 15
H.strumosus 20 12 2-354 20
H.laevigatus 7 7 7-91 8
H.glaucophyllus 1 1 38 1
H.giganteus 16 15 1-1800 19
H.eggertii 2 2 1-9 1
H.hirsutus 4 3 3-280 2
H.californicus 1 1 1 1
H.resinosus 2 2 125, 4858 2
H.silphioides 1 1 13 1
H.atrorubens 1 1 2 1
H.microcephalus 2 2 28, 300 2
H.smithii 2 2 13, 90 2
H.salicifolius 1 1 10 1
Low auto fertility in wild species makes the collection maintenance difficult. The method
of inflorescence isolation with paper bags was used at the beginning for seed production, than
the method of transferring pollen from one inflorescence to another (pollen mix), while in the
recent period a method of isolation with cages and the use of solitary bees is applied. Low
seed viability is a problem in collection maintenance, especially in perennial wild sunflower
species. Different methods for germination enhancement were tested and the results showed
that the most efficient was by removing the seed hull and seed coat (Atlagi et al., 2006).
The species in the collection have been measured and observed according to IBPGR
descriptors. The obtained results indicated that extremely high variability for a number of
characteristics existed not only between the species but also among accession within a single
species (Dozet et al., 1993; Atlagi et al., 1999; Miljanovi et al., 2000). The morphological
variability found by Miljanovi et al. (2000) was confirmed by molecular analysis in the two
taxonomically close species, H. maximiliani and H. giganteus (Safti - Pankovi et al., 2005),
which were frequently used in breeding programs. The importance of this high variability was
demonstrated through cross compatibility when crossing accessions of the same species with
cultivated sunflower. Despite the problems in maintaining the collection, the fact that all
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Jovanka Atlagi and Sreten Terzi 104
annual and 14 perennial species have been crossed with cultivated sunflower by the
conventional hybridization method (Atlagi, 2004) should be considered as success. Several
thousand crossings were made as a part of the Novi Sad breeding program, resulting in
successful transfer of desirable characteristics into cultivated sunflower.
According to Atlagi et al. (2012a) F
1
interspecific hybrids derived from hybridization
between perennial species and cultivated sunflower can be classified as genetic resources.
Perennial species reproduce vegetatively (rhizomes, tubers) so that it is relatively easy to
maintain them in ex situ collections in the field. Hybrid plants between perennial species
(H.tuberosus, H.rigidus, H.eggerttii, H.resinosus, H.strumosus, H.laevigatus, H.decapetalus,
H.divericatus and H.salicifolius) and cultivated sunflower keep the ability of vegetative
reproduction in F
1
generation. Feasibility of maintaining and using F
1
interspecific hybrids
was investigated in the collection of wild sunflower species in Novi Sad. Interspecific hybrids
were obtained using conventional crossing method in the period from 1987 to 2005 and are
grown in a quarantine field under the same conditions as wild perennial sunflower species.
The obtained results indicate that it is possible to maintain interspecific hybrids in F
1
generation in the field for 6-24 years after hybridization under the same growing conditions
(clone maintenance) as wild perennial sunflower species.
Considering difficulties in obtaining hybrids between perennial species and cultivated
sunflower, it is very useful to keep the existing hybrids and use them for obtaining further
crossing generations (BC
1
F
1
, BC
2
F
1
...).
Germplasm Evaluation and Use
Application of interspecific hybridization in cultivated sunflower breeding has an
important role in the creation of resistant hybrids to economically important diseases, which
are at present one of the main problems limiting sunflower yield. Sources of resistance are
most often found in perennial wild species. Traits that are usually associated with wild
species are resistance to pathogens and insects, but also quality and quantity of oil and
proteins (Jan and Seiler, 2007).
Considering resistance genes for sunflower pathogens, early reports date from 1970s and
describe the use of annual wild species H. annuus, H. petiolaris and H. praecox as a source of
resistance to Verticilium wilt (Verticilium dahliae Kleb.) (Hoes et al., 1973). Wild perennial
species H. resinosus, H. tuberosus, H. decapetalus, H. grosseserratus, H. nuttallii and H.
pauciflorus were found to be the source of resistance to Sclerotinia head rot (Pustovoit and
Gubin, 1974). Tolerance to Sclerotinia head rot (Sclerotinia sclerotiorum (Bk) De Bary) was
investigated in inbred lines of sunflower created within the program of interspecific
hybridization (Dedi et al., 2008), and crosses were carried out by conventional methods and
in laboratory (Vasi et al., 2002). Sclerotinia mid-stalk rot and root rot was investigated by
kori (1987) who found H. resinosus and H. tuberosus to have tolerance.
Annual wild species H. annuus, H. petiolaris and H. praecox are also a source of
resistance genes for downy mildew (Plasmopara halstedii (Farl.) Berl and DeToni) and rust
(Puccinia helianthi Schwein) (Tan et al., 1992; Quresh et al., 1993). We tested the resistance
of wild species to downy mildew and started hybridization program for transferring genes
into cultivated sunflower lines (Terzi et al., 2007).
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One factor that hampers the cultivation of sunflower is a root parasite broomrape
(Orobanche cumana Wallr). Resistance to broomrape has been found in most of the wild
perennial species (Fernandez-Martinez et al., 2000). Species of the Helianthus genus were
tested for resistance to the parasitic flowering and the possibility to transfer resistance genes
into cultivated sunflower (Terzi et al., 2010). Material testing also led to improved methods
of testing (Dedi et al., 2011).
Phoma black stem (Phoma macdonaldii Boerma) resistance was found in perennial
species H. decapetalus, H. eggertii, H. hirsutus, H. resinosus and H. tuberosus (kori,
1985). When working on the transfer of resistance to the phomopsis stem canker (Phomopsis
helianthi Munt-Cvet. et al.) embryo rescue technique was used in order to obtain interspecific
hybrids (Dozet et al., 1996) while resistance was found in perennials H. maximiliani, H.
pauciflorus, H. hirsutus, H. resinosus, H. mollis, and H. tuberosus (kori, 1985; Dozet,
1990). H. tuberosus was examined for resistance to the Phomopsis stem canker as a trait of
interest for its cultivation, but also as a source of resistance in breeding of cultivated
sunflower (Terzi et al., 2011).
Alternaria leaf spot (Alternaria helianthi (Hansf.) Tubaki and Nishihara) resistance was
observed in perennials H. hirsutus, H. pauciflorus, and H. tuberosus (Morris et al., 1983).
Rhizopus head rot (Rhizopus arrhizus Fischer) resistance was observed in perennials H.
divaricatus, H. hirsutus, H. resinosus, and H. laetiflorus (Yang et al., 1980). Powdery
mildew (Erysiphe cichoracearum DC. ex. Meret) resistance was observed in annuals H.
debilis subsp. debilis, H. bolanderi, and H. praecox (Saliman et al., 1982; Jan and Chandler,
1985) and perennials H. decapetalus, H.divaricatus, and H. laevigatus (Dedi et al., 2012).
Investigations on insect resistance genes were mostly performed in the US where insect
related yield losses are most abundant. Tolerance to sunflower moth (Homoeosoma electellum
(Hulst)) (Rogers et al., 1984), stem weevil [Cylindrocopturus adspersus (LeConte)] (Rogers
and Seiler, 1985) and sunflower beetle [Zygogramma exclamationis (Fabricius)] (Rogers and
Thompson, 1980) were mostly found in perennial species like H. tuberosus.
Oil content is higher in cultivated sunflower than in the wild species but wild species can
be used for changing the fatty acid composition affecting the oil quality (Seiler, 1998). As
reported by Pustovoit and Krasnokutskaya (1975) protein concentrations ranged from 290 to
350 g/kg in a survey of 39 wild Helianthus species which implies that there is sufficient
variability in the genus for breeding on protein concentration increase.
To reduce the cost of cultivation of commercial hybrids, it is essential to have resistance
to total herbicides such as imazethapyr and imazamox (Miller and Al-Khatib, 2002) or
sulfonylurea (tribenuron) (Miller and Al-Khatib, 2004; Terzi and Atlagi, 2008).
The accumulation of evaluation data increases the value of germplasm collections and
makes it possible for researchers and breeders to work more efficiently by selecting the right
material for their program. One example of providing efficient means for identifying useful
genetic traits is the assembly of core collections (Brothers and Miller, 1999). They are made
based on the evaluation and passport data and enable the researchers to sample the available
diversity without testing the whole collection.
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Jovanka Atlagi and Sreten Terzi 106
CYTOGENETICS
Cytogenetic studies in sunflower have a century long tradition. The first study was related
to determination of the number of chromosomes in cultivated sunflower. Karyotype of
species in the genus Helianthus was then made. The number and characteristics of
chromosomes were used to classify species and study phylogenetic relationships in the genus
Helianthus. The use of interspecific hybridization and gene transfer from wild species into
cultivated sunflower required the use of cytogenetic methods. The development of hybrid
sunflower during seventies was accompanied by studies of CMS and Rf whose application
was of great importance in the new breeding programs. Cytogenetic studies followed the
application of in vitro methods of cultivation, especially anther culture, then the study of
fertilization process in terms of separating prior- and post-zygotic incompatibility in
interspecific hybridization and determining cross-compatibility in selected hybrid parental
pairs. In recent years, cytogenetic studies are often combined with the application of
molecular markers.
The Sunflower Genome
Chromosome number in somatic cells of the cultivated sunflower (2n=34) was
determined by Tahara (1915) and confirmed by Wagner (1932), evenko (1936), and
Kostoff (1939). Studying the chromosome number in different Helianthus species, Geisler
(1931) found species with n=17, 34 and 51 chromosomes. This finding was later on
corroborated by Heiser and Smith (1955) and Georgieva-Todorova (1976). The basic
chromosome number in the genus Helianthus is n=17, while the genus is a polyploidy
complex composed of diploid (2n=2x=34), tetraploid (2n=4x=68) and hexaploid
(2n=6x=102) species (Figure 1).
Figure 1. Polyploidy in the genus Helianthus: a) H. annuus, n=34, b) H. hirsutus, n=68 and c) H.
rigidus, n=102. (Atlagi, 2004)
All 14 annual species are diploid; the 37 perennials include 25 diploid, 3 tetraploid, 6
hexaploid and 3 "mixoploid" species. H. ciliaris and H. strumosus occur in the tetraploid and
hexaploid forms, H. decapetalus in diploid and tetraploid forms (Schilling and Heiser, 1981).
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Atlagi et al. (1992) found that the diploid species H. smithii occurs also in the hexaploid
form, while the species H. strumosus occurs in diploid, tetraploid and hexaploid forms.
Most authors used to think that the basic chromosome number (n=17) comprised the
sunflower genome. Hypotheses have been made on the origin of polyploidy, whether it was
auto- or allopolyploidy. Some authors reported finding aneuploids following hybridization.
Although the annual diploids and the perennial diploids have the same chromosome number,
they are either difficult to cross or cannot be crossed at all. Heiser and Smith (1964)
concluded that these two groups of species had different genomes. Georgieva-Todorova
(1976) arrived at a similar conclusion on the basis of an analysis of interspecific hybrids. The
genome of the annual wild species evidently differs from that of the cultivated sunflower.
Analyzing the meiosis in a group of annual diploids and their interspecific hybrids, Chandler
et al. (1986) concluded that the basic chromosome number is not a single genome, i.e., that
the 17 chromosomes do not have the same origin. Thus they confirmed the finding of
Kulshreshtha and Gupta (1979) who proposed that the basic number of chromosomes in the
genus Helianthus had developed secondarily, by hybridization. Which are the original species
that had hybridized in order to give rise to the diploid sunflower species?
This question could not be answered because of the impossibility to mutually cross the
species of the genus Helianthus (cross incompatibility) and the flaws in the cytogenetic
methods (analysis of meiosis, C-bending and in situ hybridization). Great hopes have been
invested in the method of molecular markers. Using the RAPD technique, Sossey-Alaoui et
al. (1998) analyzed 40 Helianthus taxa, 36 identified and 4 non-classified. The analysis
showed that there existed the following genomes:
1 C-genome, common for all species from the three analyzed sections,
2 H-genome, specific for section Helianthus,
3 P-genome, common for perennial species (sections Atrorubens and Ciliares), and
4 A-genome, specific for section Atrorubens.
The genomic constitution was therefore HC for section Helianthus, CPA for section
Atrorubens and CP for section Ciliares. The question remains if it is possible or not to find
RAPD fragments which define the genome. It would also be important, when identifying the
different genomes, to find an effective method of comparison of RAPDs against other
molecular markers.
DNA Content
Because of the different ploidy levels in the different Helianthus species, it was
considered worthwhile to determine their total DNA contents. A study of DNA content in 22
Helianthus species and subspecies indicated that 2C DNA increased continuously from 6.4 pg
in H. neglectus to 12.02 pg in H. angustifolius (Sims and Price, 1985). The highest DNA
contents were found in H. divaricatus and H. agrestis, 16.90 pg and 25.91 pg, respectively.
Such differences are difficult to explain without considering the polyploidization. It has
been noticed that DNA content was more similar among close species than among distant
ones. This was an indication of small intraspecific variation. It was also an indication that
DNA content depended on chromosome size. So, diploid perennials have a higher DNA
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Jovanka Atlagi and Sreten Terzi 108
content than diploid annuals or some diploid species have a higher DNA content than
polyploid ones. It means that the origin of polyploid levels in the Helianthus species cannot
be proved experimentally using DNA content.
Karyotype
Karyotype describes the haploid chromosome set of an organism, i.e., the form and
length of chromosomes, length index of chromosome arms, position of the centromere and
secondary constrictions and the size and position of heterochromatic knots. The analysis of
karyotype is most frequently performed on mitotic metaphase chromosomes. Using the
classical Feulgen technique Klimokina (1940) performed a detailed karyological analysis of
H. annuus. Based on the position of the centromere, she divided chromosomes into four
groups according to their morphology. Numerous authors have conducted the analysis of
karyotype in sunflower. The nomenclature used is based on the relations between
chromosome arms. This classification distinguishes metacentric, submetacentric,
subtelocentric and telocentric chromosomes in which the ratios longer vs. shorter arm are 1.0-
1.7, 1.7-3.0, 3.0-7.0 and 7.0-?, respectively.
The karyotype of the species H. mollis was analyzed by Georgieva-Todorova et al.
(1974), H. annuus and H. debilis by Raicu et al.(1976), cultivated H. annuus by Al-Allaf and
Godward (1977), H. salicifolius by Georgieva-Todorova and Lakova (1978), H. hirsutus and
H. decapetalus by Georgieva-Todorova and Bohorova (1979), the hybrid H. annuus H.
hirsutus by Georgieva-Todorova and Bohorova (1980). Finally, Kulshreshtha and Gupta
(1981) analyzed the karyotypes of 12 H. species.
Raicu et al. (1976) found that the total length of the haploid chromosome set of the
cultivated sunflower (the cultivar Record) was 73.82 m. The lengths of the individual
chromosomes varied from 3.76 to 5.15 m. The karyotype consisted of 10 metacentrics, 3
submetacentrics and 4 subtelocentrics. Three chromosomes had secondary constrictions and a
large variation of arm ratio, from 1.08 to 5.34. In H. debilis, the length of the haploid
chromosome set was 110.19 m and the lengths of the individual chromosomes varied from
5.69 to 7.91 m. Regarding their morphology, two of them were satellite chromosomes, 6
were meta centrics, 7 were submetacentrics and 2 were subtelocentrics (Georgieva-Todorova,
1976). The karyotype of H. mollis differed from those of H. annuus and H. debilis
(Georgieva-Todorova et al., 1974). The chromosomes were short, from 3.16 to 4.50 m, and
the karyotype formula was 2SAT + 11SM + 4ST. H. salicifolius was similar to H. mollis
(Georgieva-Todorova and Lakova, 1978).
The closely related tetraploid species, H. decapetalus and H. hirsutus, had similar
karyotypes, but the former had somewhat longer chromosomes. Both species had 4 SAT
chromosomes. Georgieva-Todorova and Bohorova (1980) presented the karyotype and
ideogram of the F
1
interspecific hybrid H. annuus (2n=34) H. hirsutus (2n=68). The
somatic cells of the hybrid contained 51 chromosomes, the karyotype formula was 3
SAT+8M+11SM+4ST, and one chromosome was incomplete. The authors compared the
karyotypes of the parent species with the karyotype of the F
1
hybrid. The total length of the
chromosome set in the hybrid was 131.68 m, while H. annuus and H. hirsutus had the
lengths of 104.68 m and 172.03 m, respectively. It was difficult to identify chromosomes
of the parent species on the basis of the karyotype of the F
1
hybrid. Kulshreshtha and Gupta
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(1981) constructed the karyotypes of 12 Helianthus species. They identified only one SAT
chromosome in each of the diploid H. tuberosus species and three SAT chromosomes in the
hexaploid H. tuberosus.
Karyotypic characteristics of individual Helianthus species are useful for the study of
interspecific relations as well as for the study of evolutionary changes. The relatively small
size and the large number of chromosomes in Helianthus species make it difficult to
distinguish similar chromosomes on the basis of mitotic metaphase alone. Karyotype may be
studied on the basis of meiotic pachytene chromosomes using C- or N- bending techniques.
These techniques have allowed the identification of trisomics in many plant species (corn,
tomato, rice, barley, and others).
Because of the limited possibilities to study karyotype exclusively by cytogenetic
methods, a new direction of study has evolved, called molecular cytogenetic. Such studies
allow us to understand the genomic organization of species with both large and small
genomes. "In situ" hybridization, one of the methods employed in the cytogenetic-molecular
studies, contributes the identification of chromosomes and their arrangement which are
indicators of the evolutionary history of the genome (Heslop - Harrison, 1995). Chromosomal
variability in H. annuus var. macrocarpus was determined on the basis of heterochromatin
distribution, number and position of NORs (Nuclear Organizer Regions) and the number and
location of rDNA sequences using the method of Feulgen staining, C-bending,
fluorochromium staining, silver staining and "in situ" hybridization (Cuellar et al., 1996).
Such complex studies using chromosome markers permit:
determining whether a species is diploid, tetraploid or hexaploid, i.e., is the basic
chromosome number n=17 maybe of polyploid origin (Chandler et al., 1986);
determining whether the different Helianthus races are or are not chromosomally
different, i.e., whether they had been obtained from crosses between perennials and
annuals or between diploids and polyploids (Kulshreshtha and Gupta, 1979), and
gaining cytogenetic information from the analysis of meiotic configurations in
interspecific hybrids. Such information is exceedingly important in interspecific
hybridization.
Meiosis - Reduction Division
Meiosis or reduction division is a process of micro- and macrosporogenesis taking place
in plant stamens and ovaries, respectively. Microsporogenesis involves the development of
pollen grains or male gametes in the anther. Macrosporogenesis involves the development of
embryos or female gametes in the embryo sac.
Microsporogenesis
In Helianthus species, microsporogenesis is studied in immature anthers, most frequently
by the acetocarmine method (Georgieva-Todorova, 1976). Analyzing the meiosis in different
Helianthus species, Atlagi (1989), and Atlagi et al. (2010) made the following
observations:
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Jovanka Atlagi and Sreten Terzi 110
1 Leptonema and zygonema, early stages of prophase I, cannot be detected. Although
pachynema occurs frequently in preparations, it is not suitable for analysis because
sunflower chromosomes are thin and long. In diplonema, the chromosomes are short.
Diakinesis is the most suitable stage within prophase I for determination of the
numbers of bivalents, univalents, multivalents as well as chiasmata. Chromosome
configurations may also be determined in metaphase I, when chromosomes are
aligned along the equatorial plane. Anaphase I and telophase I occur frequently in
preparations. Meiosis II, metaphase II and anaphase II can seldom be detected in the
cultivated sunflower and almost never in the wild species, while telophase II is
frequent in both. After telophase II, tetrads are most frequently formed, although
diads, triads and pentads can be seen. Microspores are further divided mitotically,
giving rise to pollen grains (Figure 2).
Figure 2. Phases of meiosis (normal) a) Pachyten; b) Diakinesis; c) Metaphase I; d) Anaphase I;
e) Telophase I; f) Metephase II; g) Telophase II; h) Tetrads; i) Mononuclear microspores; j,k)
Microspores, l) Pollen grains. (Atlagi et al., 2010)
2 Each sunflower head contains a large number of disk flowers which differ in age
from the periphery to the center of the head. This is why a bud contains all phases of
meiosis - microsporogenesis. Each disk flower has 5 anthers, which allows one to
find all meiotic phases in a single preparation. In sunflower, the meiotic division is
asynchronous, i.e., karyokines is not followed by cytokinesis.
Georgieva-Todorova (1976) made a detailed analysis of meiosis in the cultivated
sunflower. At diakinesis, she observed 17 bivalents. The numbers of chiasmata per cell and
per bivalent were 23.88 and 14, respectively. The number of ring bivalents ranged between 3
and 10. Meiotic abnormalities were observed even in normal fertile plants, but in less than 5%
of meiocytes. These occurred as a result of spontaneous interruptions and changes.
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The analyses of meiosis microsporogenesis in interspecific hybrids (F
1
, BC
1
) are
important for determination of phylogenetic relations among Helianthus species (Figure 3).
The analysis of meiosis provides valuable data on the following:
chromosome homology and translocations (configurations at diakinesis);
changes in genetic material (number of chiasmata);
unpaired chromosomes (univalents);
non-included chromosomes (fast and lagging chromosomes);
inversions (chromosome bridges and fragments).
Figure 3. Phases of meiosis (irregular) a) Diakinesis with quadrivalent and hexavalent.; b) Metaphase I
with fast chromosomes; c) Anaphase I with lagging chromosomes; d) Anaphase I with chromosome
bridge; e) Telophase II with lagging chromosomes; f) Telophase II with chromosome bridges. (Atlagi
et al., 2010)
Pollen Viability
Mature pollen grains are yellow-orange, spherical, covered with spines (echinate), and
have three apertures. Whelan (1978) screened sunflower pollen grains by electron
microscopy. In polar view, the grains show three equidistant colpi in the wall. The equatorial
view shows that each colpus extends almost from pole to pole, with an aperture near the
middle. The germinating pollen tube emerges from one of these apertures. The diameter of
the body of the pollen grain, without the spines, varies from 33 to 39 . The abortive pollen
grain is smaller and it has an increased number of spines. Gundaev (1971) reported that the
diameter of sunflower pollen grains varied from 30 to 45 .
Preparations that are used for pollen viability determination can also be used to analyze
other pollen grain traits (sizelength, width; pore number) using light microscope Axiovert
40C (20x/0.30Ph1) Carl Zeiss Jena. Microphotographs were obtained using a digital camera
Canon Power shot A80, and the image analysis software AxioVision LE; Rel.4.3. Pollen
grain size of cultivated sunflower was in the range of 29-34m and 25-34m in wild
sunflower species (Atlagi et al., 2012b).
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Jovanka Atlagi and Sreten Terzi 112
Pollen viability is an important biological trait. It is typically assessed by staining
methods (Georgieva-Todorova, 1976; Alexander, 1969; Atlagi et al., 2012b) (Figure 4).
Figure 4. Sunflower pollen grain staining using Alexander method (Fertile-red and sterile-green pollen).
(Atlagi, 2004)
Pollen grain germination and pollen tube growth in sunflower are analyzed by fluorescent
microscopy (Xanthopoulos, 1991; Atlagi et al., 2012b) (Figure 5).
Figure 5. a. Pollen grain germination on stigma (light microscope); b. Pollen germination on stigma
(fluorescent microscope); c. Pollen tube growth through the style (fluorescent microscope). (Atlagi et
al., 2012b)
Male Sterility
The sunflower is known to possess two types of male sterility, nuclear (NMS) and
cytoplasmic (CMS). Generally, nuclear male sterility results from the action of individual
recessive gene pairs. The number of genes determining this trait differs (ms1 to ms5,
Vranceanu, 1970; ms6 to ms9, Jan, 1992). The cytology of NMS lines has not been described
in too much detail. Paun (1974) examined the NMS lines AS-110 and AS-116, which
contained the ms1 gene and which behaved similarly. He found that 2-4 univalents occur in
diplonema-diakinesis as a consequence to asynapsis or desynapsis. The author noted unequal
segregation, equatorial division and the elimination of univalents in subsequent phases.
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Chromosome clumping and agglutination were observed together with chromosome bridges
and fragments. The tetrad stage was mostly abnormal and it contained micronuclei. During
flowering, 4-6% of the pollen grains were stained with acetocarmine, but only 75% of these
had normal size and they lacked the spiny exine characteristic for Helianthus species. A
question arises as to the viability of these 75% pollen grains. Numerous authors (Nakashima
and Hosokawa, 1974; Pirev, 1968; Georgieva-Todorova, 1974) that have studied NMS found
that meiosis was normal until the tetrad stage, degenerations starting to occur at the stages of
microspores or pollen grains.
Leclercq (1969) discovered the first source of CMS in H. petiolaris ssp. petiolaris. This
discovery, together with the discovery of fertility restoration genes (Enns et al., 1970;
Kinman, 1970; Vranceanu and Stoenescu, 1971), made possible the development of hybrid
sunflower. Numerous CMS sources have been discovered in programs of crossing between
wild Helianthus species and the cultivated sunflower. Initially, the FAO list had registered 26
CMS sources (Serieys, 1991). Jan (1997) reviewed 38 CMS sources, which had been
mentioned in publications released in the period 1972-1994. These sources typically belonged
to the annual species H. annuus, H. petiolaris and H. argophyllus. Serieys (2002) reported
that the most recent FAO list included 70 CMS sources. The list specified the origin,
collection number of donor and author of the source. Of these 70 CMS sources, 62 were
derived from annual species in the section Helianthus (38 from H. annuus, 24 from other
annual species), and 8 were derived from perennial species in the section Atrorubens.
Restorer genes have been found for most of these CMS sources. CMS in the sunflower is
most frequently alloplasmatic. This term was coined by Pearson (1981) who used it to
describe male sterility resulting from interspecific and intergeneric crosses. He believed that
alloplasmatic CMS was a result of incompatibility between the nucleus and cytoplasm.
Considering the origin of CMS and the expression of this trait after incorporation into
different sunflower genotypes, cytogenetic studies in this field are both interesting and
valuable for breeding. Paun (1974) studied Leclercq's CMS. He analyzed meiosis in 4 sterile
lines and their fertile analogues. Meiosis was normal in the fertile lines, while degeneration of
sporogenous tissue occurred in the sterile lines. Degeneration occurred in pre-meiotic stages
in two sterile lines and after tetrad stage in the other two lines. The author hypothesized that
the degeneration was due to enzymatic reactions which inactivated the mechanisms for pollen
development. Studying microsporogenesis in sunflower male sterile lines, Rjabota (1969)
registered the occurrence of chromosome bridges in anaphase I and degenerative changes in
the post-meiotic cycle, i.e., in uninuclear microspore, binuclear microspore and pollen grain
stages.
Whelan and Dedio (1980) substituted H. annuus nuclei into H. petiolaris cytoplasm.
Applying a series of backcrossing, they obtained progenies whose anthers were either empty
or contained non-functional pollen. "In vitro" testing of pollen fertility in 23 BC
5
plants
showed that complete male sterility existed in 14 plants. Female fertility remained intact, as
demonstrated by normal seed set. Meiosis was normal in the BC
5
plants. Meiotic
abnormalities observed in the F
1
interspecific hybrids (univalents, chromosome bridges and
fragments) were eliminated by backcrossing, resulting in the BC
5
generation being
cytoplasmic male sterile.
Similar cytogenetic results were found when substituting H. annuus into H. maximiliani
cytoplasm (Whelan and Dorell, 1980). Meiotic abnormalities (multivalents and chromosome
bridges) were observed in early generations of crossing. The F
1
generation was observed to
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Jovanka Atlagi and Sreten Terzi 114
contain aneuploids (trisomic plants) which, together with the plants with modified anthers,
were the most frequent CMS sources. A high percentage of abnormalities in the interspecific
hybrid H. giganteus H. annuus suggested that the parents differed in genomic structure,
although they had the same number of chromosomes (Whelan, 1978).
Based on the analysis of meiosis in this F
1
interspecific hybrid, Whelan concluded that H.
giganteus differed from H. annuus in three translocations and one paracentric inversion.
Whelan concluded that the consequence of these changes in chromosome structure was the
occurrence of sterile plants. Backcrossing eliminated abnormalities, but sterility remained.
Whelan (1980) inferred that nuclear and cytoplasmatic factors are intermingled in early
generations of interspecific crossing. Since meiotic abnormalities (nuclear factors) are
eliminated by backcrossing, the male sterility remaining after BC
4
is inevitably cytoplasmic
which produces normal fertile progeny when crossed with restorers.
Using light and electron microscopy, Horner (1977) compared microsporogenesis in a
fertile line HA232 to its sterile analogue which contained CMS-PET-1. He analyzed anthers,
sporogenous tissue and chromosomes. He divided microsporogenesis into 11 stages, stages 1
to 4 ranging from the first sporogenous tissue to the initiation of tetrads, and stages 5 to 11
from late tetrad to mature pollen. Sterile and fertile analogues did not differ in
microsporogenesis until stage 5. The elongation and degeneration of tapetal cells at the end of
stage 5 caused degeneration of microspores in the tetrads, which ultimately resulted in
sterility. Similar results were obtained by Vlkova and Kovaik (1981) and Szabo et al.
(1984). Atlagi et al. (1996) studied the stability of 5 CMS sources (PET-1, PET-2, MAX-1,
GIG-1, ANN-6) during substitution into the inbred line HA89, and the stability of 5 CMS
sources (PET-1, PET-2, ANN-5, ANN-44, ANN-164) during substitution into the inbreeds L-
1, L-98, L-74 and L-22. It was found that the anthers differed in development pattern from
normal to rudimentary. Microsporogenesis developed normally until the tetrad stage in most
of the cases. Some anthers contained deformed and sterile pollen grains. The authors
concluded that the sources GIG-1 and PET-2 were unstable - their pollen viability was
10.42% and 1% to 63.43%, respectively). Atlagi and Marinkovi (1998) conducted a
cytogenetic study on potential CMS sources (interspecific hybrids with 6 H. annuus
accessions and one H. petiolaris accession). All plants in the BC
1
F
1
generation were male
sterile. Differences existed in the stage of anther development and related meiotic phases.
APPLICATION OF WILD SPECIES IN SUNFLOWER BREEDING
Wild species were first crossed with cultivated sunflower in a classical fashion by
moving pollen from male to female parent, until the embryo culture method (Chandler and
Beard, 1983) was described which facilitated obtaining more difficult cross combinations
besides other techniques like chromosome doubling (Jan and Chandler, 1989). Wild H.
annuus was also used as an intermediate parent to produce hybrids with H. giganteus and H.
maximiliani by Whelan (1978).
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Diploid Species
The annual wild species were studied in considerable detail by Chandler et al. (1986).
They found that all annuals were crossable both mutually and with the cultivated sunflower.
However, they frequently had to resort to embryo culture. The analyses of meiosis and pollen
viability, which included all annuals and their F
1
interspecific hybrids, showed that the
annuals differed in 0 to 6 translocations and 0 to 8 paracentric inversions. This was a further
proof that the basic chromosome number (n=17) is not a single genome.
Annual species are phylogenetically closer to cultivated sunflower so that they can be
used in interspecific programs without larger effort. Species H.annuus, H.argophyllus,
H.petiolaris, H.praecox (Figure 6), H.debilis, H.neglectus and H.niveus were successfully
crossed with the cultivated sunflower lines (Atlagi, 1986, 1990; kori et al.; 1988, Terzi,
2006). The obtained interspecific hybrids of different cross generations (F
1
, BC
1
F
1
BC
4
F
1
)
were most often used as sources of CMS and Rf genes.
Figure 6. Wild diploid sunflower species H.praecox (2n=2x=34) and their F
1
interspecific hybrid with
cultivated sunflower. (Atlagi, 1986)
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Meiotic abnormalities and reduced pollen viability in F
1
hybrids between wild annual
species and the cultivated sunflower have been reported by Heiser (1947, 1961), Heiser et al.
(1969), Georgieva-Todorova (1976, 1990), Whelan (1979), Atlagi (1988, 1990), and Terzi
(2006). One of the truly useful interspecific hybrids was made by Leclercq (1969) between H.
petiolaris and the cultivated sunflower. This was the first stable source of CMS (PET1) which
is still used exclusively for the development of commercial hybrids. Study of wild annuals
has lagged in recent years. Wild annuals are seldom used in inferspecific hybridization
programs because they are as sensitive to major diseases as the cultivated sunflower.
Figure 7. Wild diploid sunflower species H.salicifolius (2n=2x=34) and their F
1
interspecific hybrid
with cultivated sunflower. (Atlagi, 2004)
Wild perennial sunflowers of various ploidy levels have been mutually crossed mostly for
the purpose of cytotaxonomy (Heiser and Smith, 1955; Jackson, 1963; Heiser et al., 1969;
etc.). Wild perennials were also crossed with the cultivated sunflower (Christov, 1991), but
these interspecific hybrids were seldom subject to cytogenetic analysis. Diploid perennials are
interesting for breeders as potential sources of resistance to diseases (H. giganteus, H.
maximiliani), high oil content in seed (H. salicifolius) (Figure 7) or development of a new
ideotype (H. mollis). Crossability between diploid perennials and the cultivated sunflower is
poor, as demonstrated in studies of Georgieva-Todorova (1976, 1990), Jan (1987), Atlagi
(1994a), Atlagi et al. (1995), etc.
Using the embryo culture method, Kruter et al. (1991) succeeded in obtaining hybrids
between H. mollis and H. maximiliani on one hand and the cultivated sunflower on the other.
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Cytogenetic analyses of F
1
interspecific hybrids (Georgieva-Todorova, 1967, 1976, 1990;
Whelan, 1978; Atlagi et al., 1995) detected numerous meiotic abnormalities (uni- and
quadrivalents in diakinesis, dislocated chromosomes in meta-, ana- and telophases, and
chromosome bridges in anaphase I). Also, these authors found complete sterility or reduced
pollen viability in F
1
hybrids. These results indicated that the genomes of diploid perennials
and diploid annuals differ (Heiser and Smith, 1964), which limits the use of the former in
breeding programs.
Tetraploid Species
The analyses of meiosis and pollen viability in the tetraploid species has raised the
question of the origin of polyploidy in the sunflower. Georgieva-Todorova and Bohorova
(1979) and Georgieva-Todorova (1990) reported that meiotic abnormalities and reduced
pollen viability in tetraploid species H. hirsutus, H. decapetalus, H. strumosus and H.
scaberimus reflect their allopolyploid nature. On the other hand, Atlagi (1991) reported that
these species had regular meiosis, which suggests autopolyploidy.
Hybridization between tetraploids and the cultivated sunflower was observed only in a
few cases, by Heiser et al. (1962), Georgieva-Todorova et al. (1979), Pustovoit (1975),
Christov (1991) and Atlagi (1994b). Georgieva-Todorova et al. (1979), Georgieva-Todorova
(1984), Atlagi (1994b), and Terzi (2006) have successfully crossed the tetraploid species H.
hirsutus (Figure 8), H. decapetalus, H. laevigatus and H. strumosus with the cultivated
sunflower and conducted cytogenetic analyses of the hybrids.
Figure 8. Wild tetraploid sunflower species H.hirsutus (2n=4x=68) and their F
1
interspecific hybrid
with cultivated sunflower. (Atlagi, 2004)
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Jovanka Atlagi and Sreten Terzi 118
The results of these analyses showed an exceedingly high percentage of meiotic
abnormalities and a frequent occurrence of complete sterility. Obviously, it is difficult to
transfer desirable genes from these species into the cultivated sunflower. In order to make
these crosses, conventional hybridization methods have to be combined with the embryo
rescue method (Kruter et al., 1991) and chromosome doubling in the F
1
and BC
1
interspecific hybrids (Jan, 1988).
Hexaploid Species
The species from the hexaploid group most frequently used in sunflower breeding are H.
tuberosus (a source of resistance genes to Phomopsis stem canker, Alternaria leaf spot and
downy mildew), H. pauciflorus (H. rigidus) (resistance to disease agents and high protein
content in seed) and H. resinosus (resistance to disease agents and high content of oleic acid
in seed). These species have undergone extensive cytogenetic studies by a number of
researchers. Kostoff (1934) was the first to conduct a detailed analysis of H. tuberosus and he
established two hypotheses on the genomic structure of the species. First, H. tuberosus is
autohexaploid (AAAAAA); and second, H. tuberosus is amphiploid (AABBCC) made from a
cross of an autotetraploid and a diploid form. In 1939, the same author conducted a study of a
hybrid between H. tuberosus and the cultivated sunflower, which confirmed his hypothesis on
the different genomes in these two species.
Clevenger and Heiser (1963) claimed on the basis of cytogenetic analyses that H.
tuberosus is a natural hybrid but the results of Georgieva-Todorova (1990) and Atlagi et al.
(1993) indicated that it is an original species. Many researchers studied the meiosis and pollen
viability in F
1
hybrids between H. tuberosus and the cultivated sunflower (Kostoff, 1939;
Heiser and Smith, 1964; Cauderon, 1965; Heiser et al., 1969; Pustovoit, 1969; Georgieva-
Todorova, 1990; Atlagi et al., 1993; Espinasse et al., 1995; Terzi, 2006). Their results
invariably showed that the complete sterility and reduced fertility in these interspecific
hybrids were due to a large number of meiotic abnormalities occurring as a consequence of
the differences in chromosome number and structure between the parent species.
Figure 9. Wild hexaploid sunflower species H.rigidus (2n=6x=102) and their F
1
, F
1
BC
1
interspecific
hybrids with cultivated sunflower. (Atlagi, 2004)
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Sunflower Genetic Resources 119
Table 6. The potential for interspecific hybridization of wild and cultivated sunflower
Species
Chrom
No.
1981 1991 year. 1992 - 2006 year.
Accession No. F
1
gen. Accession No. F
1
gen.
Polli-
nated
Crossed
No.
of plants
Polli-
nated
Crossed
No. of
plants
H.annuus 17 24 17 123 28 11 169
H.petiolaris 17 22 20 35 23 3 7
H.argophyllus 17 8 6 24 9 2 30
H.neglectus 17 2 2 14 4 1 12
H.debilis 17 12 9 32 13 1 3
H.praecox 17 11 9 24 19 4 0
H.niveus 17 2 1 8 3 0 0
H.mollis 17 4 3 10 7 2 0
H.salicifolius 17 2 1 7 7 2 0
H.maximiliani 17 7 3 10 31 5 0
H.occidentalis 17 3 1 8 0 0 0
H.nuttallii 17 3 1 9 24 1 0
H.smithii 17 2 2 27 0 0 0
H.decapetalus 17, 34 5 1 28 10 1 0
H.hirsutus 34 3 2 66 3 1 0
H.strumosus 34, 51 8 1 13 14 5 24
H.laevigatus 34 5 3 51 3 1 4
H.tuberosus 51 17 9 90 11 4 27
H.rigidus 51 7 3 105 11 4 0
H.eggertii 51 1 1 5 1 1 0
H.resinosus 51 2 2 89 3 1 0
H.divaricatus 17 6 1 1 13 2 3
H.giganteus 17 6 1 0 12 0 0
H.grosseserratus 17 3 0 0 16 0 0
H.microcephalus 17 2 0 0 1 0 0
H. pauciflorus (H. rigidus) is another wild species extensively used in sunflower breeding
programs (Figure 9). The species itself has not been extensively studied (Atlagi, 1996a), but
F
1
interspecific hybrids between H. rigidus and the cultivated sunflower were studied by
Whelan (1978), Georgieva-Todorova (1990) and Atlagi (1996a). All of these studies showed
irregularities in chromosome pairing and diakinesis. Of all hexaploid species, Georgieva-
Todorova (1990) found H. resinosus to be most similar to the cultivated sunflower. Within
the scope of cytogenetic analyses of interspecific hybrids, the analyses of meiosis and pollen
viability were studied by the largest number of researchers. In addition to crossability,
sterility and reduced fertility in interspecific hybrids are most indicative of the applicability of
these hybrids in sunflower breeding programs. Based on her long-term studies, Georgieva-
Todorova (1984, 1990) concluded that pollen viability is invariably associated with meiosis
as well as that it is genetically controlled. Conversely, Chandler et al. (1986) came to a
conclusion that pollen viability is invariably affected by the number and type of meiotic
abnormalities, but these effects do not necessarily have to be direct. Cytogenetic studies have
mostly been done on F
1
interspecific hybrids. However, analyses of BC
1
F
1
hybrids showed
even larger percentages of meiotic abnormalities, as well as the occurrence of aneuploids,
plants with different chromosome numbers, reduced pollen viability, etc. (Whelan, 1979;
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Jovanka Atlagi and Sreten Terzi 120
Whelan and Dorrell, 1980; Atlagi, 1996b; Atlagi and kori, 1999). Although Whelan
(1979) and Whelan and Dorrell (1980) claimed that backcrossing eliminates meiotic
abnormalities observed in F
1
interspecific hybrids, cytogenetic analyses of BC hybrids have
shown that the elimination takes place in later generations of backcrossing. Defining
problems associated with the use of wild Helianthus species in sunflower breeding programs,
Atlagi and kori (2000) pointed out that phylogenetic differences among species are as
important if not more important than differences in ploidy level.
Recent studies of interspecific hybridization in sunflower have included various aspects
of occurrence of partial hybrids in wide crosses between sunflower (H. annuus) and perennial
species (H. mollis and H. orgyalis) (Faure et al., 2002a, 2002b, 2002c).
The hybridization potential presented in table 6. shows that majority of species apropos
accessions, was crossed in the period between 1981 and 1991. That is a result of more
intensive work on interspecific program, because larger number of crosses was made than in
the following period. Similar to the Novi Sad program, large number of crosses between wild
species and the cultivated sunflower was made and the potential of interspecific hybridization
usage in sunflower breeding was described by Georgieva-Todorova, 1990, Christov, 1991,
Jan, 1997.
CONCLUSION
Based on literature data and the results of our own studies, it became clear that the
method of interspecific hybridization is extensively used in sunflower breeding programs.
Obstacles that precede interspecific hybridization are also in the forming and
maintenance of a wild species collection and making accessions available to breeders. The
most frequent barriers for interspecific hybridization application are: cross incompatibility
(prezygotic and postzygotic - embryo abortivity), lowered fertility or complete sterility of F
1
and other early generations of interspecific hybridization. Differences in ploidy, phylogenetic
origin and taxonomic belonging of the wild species in comparison to the cultivated sunflower
are the cause of the mentioned barriers.
Interspecific hybridization brings not only desirable, but also a large number of
undesirable traits (branching, small head diameter, low oil content, etc.) and that is why it is
necessary to back cross F
1
interspecific hybrids with cultivated sunflower. Cytogenetic
analysis of BCF
1
hybrids have shown large percentage of irregularities in meiosis, aneuploids,
plants with different chromosome number, lowered pollen viability. On the other hand,
desirable genes are lost after several back crosses with cultivated sunflower. That is why it is
necessary to analyze not only on cytogenetic, but also on molecular level for the presence of
wild species genome in comparison to the cultivated sunflower genome in interspecific
hybrids.
Application of wild species in cultivated sunflower breeding is the most significant in
creating resistant hybrids to economically important diseases like Phomopsis stem canker and
Sclerotinia rot and root parasite broomrape. Perennial wild species were most often the source
of resistance. Nevertheless, annual species like H.annuus and H.petiolaris provided fertility
restoration genes for CMS (PET-1) and some new sources of CMS.
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Sunflower Genetic Resources 121
Despite the difficulties in wild sunflower collection maintenance, interspecific
hybridization and isolation of desirable genes, Helianthus genus remained a constant source
of material for improvement of cultivated sunflower. Greater efficiency in wild sunflower
germplasm usage depends on successful application of modern techniques like marker-
assisted selection and a growing database on gene function and location, while having secured
germplasm collections.
REMARKS
The authors note that in the writing of the chapter they used most of the text from a
review paper Roles of interspecific hybridization and cytogenetic studies in sunflower
breeding by Atlagi J. published in the Helia journal in 2004.
ACKNOWLEDGMENTS
We are grateful to the Ministry of Education, Science and Technological Development of
Republic of Serbia, which has funded projects in which the research was conducted and
produced results partly described in this chapter, including the current project TR31025. The
authors thank the Institute of Field and Vegetable Crops.
The technical assistance of Jasminka Pilipovi and uro Kovaevi is gratefully
acknowledged for the help and enthusiasm to work on wild species maintenance, interspecific
hybridization, cytogenetic analysis and technical assistance with writing.
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas 2014 Nova Science Publishers, Inc.
Chapter 6
FUNCTIONAL GENOMICS AND TRANSGENESIS
APPLIED TO SUNFLOWER BREEDING
Sebastian Moschen
1,2,
, Laura M. Radonic
1,*
,
Guillermo F. Ehrenbolger
1,2
, Paula Fernndez
1,2,3
,
Vernica La
1,2,4
, Norma B. Paniego
1,2
,
Marisa Lpez Bilbao
1
, Ruth A. Heinz
1,2,4
and H. Esteban Hopp
,1,4
1
Instituto de Biotecnologa, Centro de Investigaciones en Ciencias
Agronmicas y Veterinarias, Instituto Nacional de Tecnologa
Agropecuaria - INTA, Hurlingham, Buenos Aires, Argentina
2
Consejo Nacional de Investigaciones Cientficas y Tcnicas,
Ciudad Autnoma de Buenos Aires, Argentina
3
Escuela de Ciencia y Tecnologa, Universidad Nacional
de San Martn, San Martn, Buenos Aires, Argentina
4
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad Autnoma de Buenos Aires, Argentina
ABSTRACT
The advances in genomics and post genomics in the last decade allowed the
discovery and functional characterization of many genes simultaneously on a genome-
wide scale. However, the sunflower genome was not systematically sequenced until the
recent advent of next-generation sequencing technologies and is still in progress. In
parallel, comprehensive EST datasets were developed and used to design oligo-
nucleotide-based microarrays focusing both in genotyping and expression analysis
purposes, which in turn, help to study the transcriptomics response to different growing
conditions as water deficit, senescence or response to pathogens. In addition, first
metabolomics analyses of tolerance to diseases started few years ago. This chapter
reviews the functional genomics analysis of several important sunflower characters
including the development and application of bioinformatic approaches to explore
SM and LMR are equal co-senior authors.
Unit for Environmental Sciences and Management,
North-West University, Potchefstroom, South Africa
ABSTRACT
Numerous insect species attack sunflower in Africa. While only a few species have
high pest status, the majority are of little importance or occur sporadically. Sunflower is
subject to insect damage from planting onwards to drying of seeds on the heads. The
pests that attack sunflower are largely polyphagous and attack a variety of crops and wild
host plants.
Pests can be categorised into seedling pests, leaf pests and pests of sunflower heads.
Seed and seedlings are mainly damaged by soil insects, while mainly Lepidoptera larvae
and Hemiptera species cause damage to sunflower heads. The most important are the
semi-looper, Trichoplusia orichalcea, a highly sporadic pest that attacks leaves of older
plants and when outbreaks occur, total defoliation may result and the false chinch bug,
Nysius natalensis which damages developing seed, resulting in a reduction in yield, oil
content and germination of damaged seeds.
The African bollworm, Helicoverpa armigera is a common pest which infests
sunflower heads during the budding stage and cause damage to achenes from anthesis
onwards. Actual damage and not number of larvae per head is the only criterion which
can be used in the determination of economic injury levels for control of African
bollworm on sunflower.
With the exception of the African bollworm, no economic threshold levels for
chemical control of sunflower pests have been developed in southern Africa. This often
leads to unnecessary application of insecticides. The management of N. natalensis and H.
armigera on sunflower is discussed in this chapter.
Keywords: Sunflower, damage, pests, Helicoverpa armigera, Nysius spp.
hannalene.duplessis@nwu.ac.za.
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Hannalene du Plessis 228
INTRODUCTION
Sunflower is utilized by many insect species, both as food plant and as a source of pollen
and nectar. A number of insect species have adapted to cultivated sunflower and have become
economically important pests (Charlet et al., 1997). Rajamohan (1976) reported 255 different
species of insects and mites attacking sunflower globally, while Misari (1990) recorded 172
species belonging to 77 families and 9 orders in Nigeria. Eighty insect species were reported
from sunflower roots, stems, leaves and flower-heads in Kenya (Khaemba & Mutinga, 1982).
Only those species regarded as of economic importance will be discussed in more detail.
PEST OF ECONOMIC IMPORTANCE
Although many insect species occur on sunflowers in southern Africa, only a few are
considered to be of potential economic importance. These are: the common cutworm [Agrotis
segetum (Denn. & Schiff) (Lepidoptera: Noctuidae)], dusty surface beetles, Gonocephalum
simplex Fab. (Coleoptera: Tenebrionidae), greater false wire worms (Somaticus spp.)
(Coleoptera: Tenebrionidae), ground weevils, Protostrophus spp. (Coleoptera:
Curculionidae), the African bollworm, Helicoverpa armigera (Hbner) (Lepidoptera:
Noctuidae), plusia looper, Trichoplusia orichalcea (Fabricius) (Lepidoptera: Noctuidae) and
the false chinch bug, Nysius natalensis Evans (Hemiptera: Orsillidae). These insects occur
sporadically in high numbers and are then injurious to sunflower crops. The pest complex
regarded as major pests in Kenya are Agrotis spp., Plusia orichalcea Fab, H. armigera and
Nezara viridula L. (Khaemba & Mutinga, 1982), which is similar to southern Africa with the
exception of N. viridula. Nysius natalensis is the most important hemipteran pest in South
Africa.
SEEDLING PESTS
Although various types of damage are caused to sunflower seedlings, the damage
symptoms are characteristic of particular pest species. Damage caused to seedlings vary from
cutting through the stems at or just below the soil surface by dusty surface beetles
(G. simplex), chewing into the stem of seedlings just below the soil surface by greater false
wire worms (Somaticus spp.) and above-ground feeding on seedlings, mainly on leaves by
ground weevils (Protostrophus spp.). These seedlings can be destroyed during severe
infestations (Du Plessis, 1999).
Cutworms (Agrotis spp.) damage seedlings by cutting through the stems at or just below
the soil surface. Severe cutworm infestations can destroy a crop and necessitate replanting.
Strategies to manage cutworms include the application of insecticides and cultural control
strategies. In areas with very cold winters, such as in southern Africa, cultural control by
means of winter cultivation which destroys winter weeds and bring the larvae and pupae to
the soil surface where they are taken by birds or the pupae killed by frost, can reduce
infestation levels present in fields. The destruction of winter weeds deprives cutworm moths
of oviposition substrates and larvae of a food source. Application of these practices over a
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Insect Pests of Sunflowers in Africa 229
wide area, results in decreased moth numbers by the beginning of spring (Drinkwater, 1997).
Preplant weed control is the best method of cutworm control. If there are no volunteer plants
on fields during spring, the large numbers of cutworm moths that start flying during early
spring cannot lay their eggs in fields. Where practically possible, all fields must be cultivated
35 days before planting, or even earlier, to keep them free from weeds until planting time.
This will also cause small cutworm larvae that may already be present in fields at that stage to
die of starvation (Drinkwater, 1997).
PEST OF SUNFLOWER ROOTS
Khaemba and Mutinga (1982) considered root-feeding pests in Kenya to be of little
economic importance since their attacks are sporadic. Termites were, however, reported by
Evans (1951) to cause considerable losses in sunflower by damaging the roots that resulted in
lodging of the plants in Tanzania. Riekert & Van den Berg (2003) evaluated 13 sunflower
cultivars in South Africa and found no plants of any of the cultivars to lodge as a result of
termite damage indicating resistance of these cultivars to termites in contrast to maize plants
that lodge under similar infestation pressures. The planting of sunflower did, however, not
suppress termite activity in the subsequent seasons (Riekert & Van den Berg, 2003).
PEST OF LEAVES
Plusia sp. was reported to visit flowering sunflower plants in South Africa (Cockerell,
1916). Trichoplusia orichalcea larvae can cause devastating damage to sunflower. If
outbreaks occur and large numbers are present, plants may be totally defoliated, stems and
flower buds are damaged, often resulting in total crop loss. Damage usually does not occur on
the edges of fields and is therefore not visible from outside the field (Du Plessis, 1999).
PESTS DURING THE PRE-HEADING AND HEADING STAGES
The most important pests of sunflower during the heading stages of crop development are
hemipterans and H. armigera. Although hemipterans have been reported in abundant numbers
on sunflower in many regions in Africa, none of them, except for Nysius natalensis, have
been studied in detail. Because of their small size, hemipterans are often inconspicuous on
sunflower heads and the qualitative damage they may incur is therefore largely
underestimated. Due to its omnipresence and because H. armigera may attain pest status
wherever sunflower is grown it has been studied on sunflower.
Hemipterans have been reported as the most abundant insects occurring on sunflower
plants during the pre-heading growth stages in Nigeria (Misari, 1990). They suck sap from
stems and leaves resulting in stunted growth of plants. Macrosteles spp. (Cicadellidae)
comprised over 90% of all these hemipterans reported in Nigeria (Misari, 1990). It has,
however, not been documented as such for other African countries. Calidea dregii Germar
(Hemiptera: Pentatomidae), C. bohemia (Stl) (Hemiptera: Pentatomidae), and Nezara
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Hannalene du Plessis 230
viridula L. (Hemiptera: Pentatomidae), are regarded as pests of immature sunflower seeds in
east Africa (Hill, 1975; Khaemba & Mutinga, 1982). Very high populations of C. dregii
(140 000/ha) was reported by Evans (1951) on sunflower in Urambo, Tanzania and their
feeding resulted in reduced seed weight, oil content and subsequent seed viability.
The intensive feeding by hemipterans during the heading stage results in deformed heads
which delay flower opening (Misari, 1990), a phenomenon which is also observed under
heavy infestation of N. viridula in South Africa (H. du Plessis, personal observation).
The most common hemipteran family on sunflower in South Africa is, however, the
Lygaeidae (seed bugs) (Du Toit & Holm, 1992). The genus Nysius was previously classified
under the family Lygaeidae. Most species of the Lygaeidae, both ground-living and plant-
living, subsist upon seeds of many plant species (Sweet, 1960). The name seed bugs was
therefore suggested for the Lygaeidae by Sweet (1964). The family Lygaeidae was, however,
divided into 11 families (Henry, 1997), with the family Orsillidae (also containing the genus
Nysius) now considered to be a distinct family from the Lygaeidae (Sweet, 2000). Misari
(1990) recorded Nysius stali Evans to occur from the heading stage to the grain stage on
sunflower in Nigeria. The occurrence of the false chinch bug, Nysius natalensis Evans
(Hemiptera: Orsillidae) is similar on sunflower in South Africa and sunflower crops are
usually damaged in late summer (Du Plessis et al., 2007). Damage by N. natalensis to seeds
results in a reduction in yield, oil content and germination of damaged seeds.
Sunflower in South Africa is grown mainly between the 24S and 30S latitudes, where
the climate is semi-arid, characterized by erratic and relatively low rainfall, low humidity and
high radiation intensity during summer (Nel, 1998). Nysius occurs throughout the South
African sunflower production area but only attains pest status in certain areas (Du Plessis et
al., 2007). In a study to determine the effect of temperature on N. natalensis development and
survival, Du Plessis et al. (2011) found that the females lay eggs sooner at higher
temperatures (shorter pre-oviposition period), but that the optimum temperature for
oviposition (most eggs laid) is between 26 and 28C and it is also the temperature range
where the female lifespan is the longest (19 28C) (Table 1). The lower threshold
temperature for N. natalensis eggs and nymphs differed by only 1.25C. Eggs will not hatch
at temperatures too low for nymphal development, which is 14.0C for eggs and 15.2C for
nymphs (Du Plessis et al., 2011). Since high summer temperatures prevail throughout the
sunflower production area of South Africa and the most favourable temperature range for N.
natalensis development was determined as 26 to 38C, the potential for rapid population
build-up exists during the sunflower production season. Potential therefore also exist that this
pest can become important on sunflower in other African countries too.
Wild host plants and crops such as grain sorghum play an important role in sustaining
populations of N. natalensis (Kruger et al., 2008; Du Plessis et al., 2007). To determine
whether a plant is a suitable host of a particular insect, the insect must be able to complete its
life cycle on the host plant and replace itself, or have an intrinsic rate of population increase
equal to or greater than zero (Zalucki et al., 1986). Shorter development times and higher
rates of reproduction with low mortality rates on a host indicate greater suitability of a
particular host plant (Awmack & Leather, 2002). Eggs, nymphs, and adults of N. natalensis
have been collected from 26 species of host plants in eight families, as well as from cultivated
sunflower plants in South Africa (Du Plessis et al., 2007).
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Table 1. Mean fecundity and longevity (+ S.E.) of Nysius natalensis females at constant temperatures and 14L:10D photoperiod.
On the day of their final moult, single male-female pairs were confined to Petri dishes (90 mm diameter)
and life history parameters recorded until females died
Temperature
( 1 C)
n Pre-
oviposition
period (days)
Post-
oviposition
period (days)
No. of days
during which
no eggs were
laid
Longevity of
adult female
(days)
Total no.
of eggs laid
Mean no. of
eggs laid per
day
Max. no. of
eggs laid per
day
Infertile
eggs (%)
19 28 13.6 + 1.3 a 0.9 + 0.2 ab 31.3 + 3.2 a 45.0 + 2.9 a 122.0 + 13.6 b 3.9 + 0.3 a 10.9 + 0.7 a 1.2 bc
26 35 5.9 + 0.4 b 1.2 + 0.3 ab 3.3 + 1.1 b 32.4 + 2.2 b 245.8 + 19.6 a 10.6 + 0.7 b 21.3 + 1.1 b 2.8 a
28 28 6.3 + 0.7 b 1.5 + 0.5 a 2.5 + 0.7 b 28.9 + 1.8 b 275.8 + 28.8 a 12.2 + 0.8 bc 23.5 + 1.5 b 0.1 c
31 37 4.3 + 0.2 c 0.6 + 0.2 ab 0.9 + 0.3 b 13.9 + 1.0 c 107.1 + 12.4 b 11.7 + 0.8 bc 22.3 + 1.3 b 4.9 ab
36 30 2.3 + 0.1 c 0.2 + 0.1 b 0.3 + 0.1 b 8.8 + 0.7 c 95.7 + 12.3 b 15.0 + 1.5 c 25.9 + 2.1 b 1.7 c
38 31 3.6 + 0.2 c 0.3 + 0.2 b 0.4 + 0.2 b 9.2 + 0.5 c 81.6 + 8.8 b 14.6 + 1.0 c 23.5 + 1.6 b 0.6 c
Means within the same column followed by the same letter do not differ significantly at P = 0.05 (Tukeys HSD). (From: Du Plessis et al., 2011).
(Reproduced with permission of the Entomological Society of Southern Africa).
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Hannalene du Plessis 232
Table 2. Plant species recorded as host plants of Nysius natalensis in South Africa.
Sampling of Nysius natalensis and host plants was conducted through a roadside
survey in each quarter-degree grid in the area in the sunflower production
area of South Africa. Nysius natalensis was reared for one generation
in the laboratory on seed of each wild host plant to confirm the host status
of the respective plants (Du Plessis et al., 2007).
Family Plant species Common name
Amaranthaceae
Amaranthus hybridus L. subsp. hybridus var.
hybridus
Cape pigweed
A. spinosus L. Thorny pigweed
A. thunbergii Moq. Red pigweed
Asteraceae Conyza aegyptiaca (L.) Aiton Fleabane
C. albida Spreng. Tall fleabane
C. bonariensis (L.) Cronq. Flax-leaf fleabane
C. canadensis (L.) Cronq. Horseweed fleabane
C. podocephala DC. Conyza
Felicia muricata (Thunb.) Nees subsp. muricata -
Helichrysum atgyrosphaerum DC. -
H. nudifolium (L.) Less -
H. rugulosum Less -
Nidorella resedifolia DC. subsp. resedifolia -
Pseudognaphalium luteo-album (L.) Hilliard &
Burtt
Jersey cudweed
P. oligandrum (DC.) Hilliard & Burtt -
Senecio consanguineus DC. Ragwort
S. inornatus DC. -
Campanulaceae Wahlenbergia undulata (L.f.) A.DC. Pale bluebell
Chenopodiaceae Chenopodium album L. White goosefoot
C. carinatum R. Br. Green goosefoot
C. cristatum F. Muell. Crested goosefoot
Poaceae
Melinis repens (Willd.) Zizka subsp. grandiflora
(Hochst.) Zizka
(Hochst.) Zizka
Natal red top
Portulacaceae Portulaca oleracea L. Pigweed
P. quadrifida L. Wild purslane
Solanaceae Physalis viscose L. Sticky gooseberry
Verbenaceae Verbena bonariensis L. Wild verbena
Reproduced with permission of the Entomological Society of Southern Africa
Fewer eggs are laid by females when they fed on stems of plants, in spite of the seed of
these plants being a suitable food source for their survival. Seeds were therefore found to be
essential for N. natalensis reproduction. The insect is polyphagous and a variety of wild host
plants as well as sunflower are suitable for feeding and reproduction. As N. natalensis adults
are good fliers, it is likely that nymph-to-adult host switching is a common phenomenon. It is
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Insect Pests of Sunflowers in Africa 233
also likely that the insects switch hosts between generations when adults migrate and lay eggs
on plant species different from those on which they developed. The effects of within-
generation host plant switching were studied by Du Plessis et al. (2012), by providing various
combinations of foods to nymphs and adults. Nymphs were reared on crushed seed of five
plant species, namely A. hybridus, P. oleracea, C. album, C. albida and H. annuus. After
completion of the nymphal stage, emerging adults of each group were either provided with
the same diet, or were transferred to different seeds as adult food. The duration of the pre-
oviposition period is affected by the nymphal food source, but fecundity is determined by
food consumed during the adult stage (Du Plessis et al., 2012). The number of eggs laid by N.
natalensis is affected by adult food as well as by the interaction between nymphal and adult
food sources while adult longevity is influenced by the host plant species consumed by both
nymphs and adults (Du Plessis et al., 2012).
The wild host plants, A. hybridus, P. oleracea, C. album, and C. albida were also
evaluated as paired combinations in two-choice experiments to determine the comparative
attractiveness of sunflower and wild host plants for oviposition by N. natalensis. The control
was a piece of pipe cleaner (Du Plessis et al., 2012). When given a choice, N. natalensis
females prefer to lay their eggs on wild host plants (weeds), compared with sunflower (Figure
1). Sunflower is therefore not the preferred host, but N. natalensis lay its eggs on sunflower if
its preferred host plants are removed or dead (Du Plessis et al., 2012). These results explain
host plant switching by N. natalensis from its wild host plants to sunflower as a result of
restricted temporal availability of wild host plants. It also explains their injuriousness to late-
planted sunflower when senescence of weed species occurs during autumn. This period often
coincides with seed fill of late-planted sunflower, providing host plants for the insect with a
high moisture content, as well as seeds necessary for reproduction of the pest. Weeding of the
headlands of sunflower during seed fill of the crop results in destruction of the preferred host
plants of N. natalensis. Insect consequently move to sunflower where they feed and cause
damage (Du Plessis et al., 2007).
The risk of short-range dispersal from host weed species should be limited to protect
sunflower crops, especially late-planted crops, from N. natalensis damage. Headlands and
adjacent fields should be kept weed free from the beginning of the season to limit pest
population build up. Furthermore, weedy headlands and adjacent fields on which weedy host
plants occur should not be hoed during seed-fill period of sunflower crops because these
activities result in destruction of the wild host plants, causing subsequent invasion of
sunflower fields (Du Plessis et al., 2007). Late plantings should therefore be avoided where
possible and particular attention should be paid to informed control of annual weeds in the
vicinity of the crop (Du Plessis et al., 2012).
The decision to apply insecticides for control of this pest should take the following into
consideration. It is a polyphagous pest that is highly mobile and continuous re-infestations
can occur. Timing of insecticide application is therefore important. Most damage to sunflower
seeds inflicted by N. natalensis is done at the distal end of seeds (Du Plessis et al., 2005). It
takes the cotyledons approximately 10 to 12 days to develop and reach the distal end of the
husk, depending on the cultivar. If damage is the only concern, it is not necessary to spray
during anthesis. Keeping in mind that anthesis commences at the periphery of the sunflower
head and progresses inward, it can be equal to the period that heads of certain cultivars take to
complete anthesis (Du Plessis et al., 2005). The period that the heads take to turn downward
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Hannalene du Plessis 234
after completion of anthesis will, however, determine the period of application of insecticides
against N. natalensis during seed fill (Du Plessis et al., 2005).
Source: Entomologia Experimentalis et Applicata 2012 The Netherlands Entomological Society
Figure 1. Mean number ( SD) of eggs laid by Nysius natalensis on various host plants (Helianthus
annuus, Amaranthus hybridus, Chenopodium album, Conyza albida, and Portulaca oleracea) in paired-
choice tests. Means of a pair capped by the same letter do not differ significantly (Tukeys HSD:
P>0.05). (Du Plessis et al., 2012).
The African bollworm, H. armigera, is an important pest of many crops in many parts of
the world (Zalucki et al., 1986; Sharma, 2001). It is also regularly present during the
reproductive stage of cultivated sunflower in Africa. Its attractiveness to sunflower is
demonstrated by its use as a trap crop in and around organic cotton fields in Tanzania (Cherry
et al. 2003). Larvae occur from the budding stage onwards (Von Maltitz, 1993). Levels of
infestation vary between localities and seasons, sporadically reaching epidemic proportions.
First and second instar larvae are mainly found on sunflower buds, with a feeding preference
for involucral bracts. Later instar larvae feed more extensively than younger larvae,
consequently doing more damage. One mature larvae feeding inside a bud may completely
destroy the immature florets. However, plants infested at the budding stage escape serious
feeding damage as the majority of larvae mature during anthesis (Von Maltitz, 1993; Du
Plessis, 1997). Larvae of all instars also burrow under and between bracts into the receptacles
(Von Maltiz, 1993), and are, therefore often not exposed to insecticides. Insecticides for H.
armigera control on sunflower in South Africa are applied aerially and the question often
arises whether effective insecticide application is limited by the downward inclination of
sunflower heads and by the fact that the heads are often turned in the opposite direction to
aerial application. Du Plessis (1997) found aerial application of insecticides for bollworm
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Insect Pests of Sunflowers in Africa 235
control to be effective during the budding as well as the anthesis and pollination stages when
later instars, which are more damaging, occurred on heads.
Simulated bollworm damage was done to florets (R-5.1stage) and young achenes in the
milky stage (R-6.0) of sunflower in a field trial in South Africa (Du Plessis, 1997). R-5.1 is
the reproductive stage at which 10 % of the head area (disk flowers) has completed or is in
flower. R-6.0 is the reproductive stage in which anthesis and pollination are completed and
the ray flowers are wilting (Schneiter and Miller, 1981). The mean mass of randomly chosen
achenes in these heads increased at increased levels of damage (Figure 2).
Compensation for lost florets and achenes, therefore, occurred over the entire head.
Compared with undamaged heads, the mean mass of achenes was significantly higher at
levels of 15, 20 and 30 % damage at both reproductive stages (Figure 2). The total mass of
fertile achenes per head did not differ between damaged and undamaged heads, up to a 30 %
level of damage (although lower at 5 and 30 % damage levels) (Figure 3) for both
reproductive stages evaluated. It showed that sunflower has the ability to compensate for
damage (Du Plessis, 1997). Sunflower variety and climatic conditions may provide results
which differ from those found by Du Plessis (1997).
Figure 2. Mean mass of 15 achenes surrounding simulated bollworm damage area(s) and chosen at
random in sunflower heads at different levels of damage done to florets in the R-5.1 reproductive stage
and to young achenes in the milky stage (R-6.0). = LSD
T
(P = 0.05). (From: Du Plessis, 1997).
(Source: African Crop Science Journal)
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Hannalene du Plessis 236
Figure 3. Mean mass of fertile achenes per sunflower head at different levels of simulated bollworm
damage (n=24) done to florets in the R-5.1reproductive stage and to young achenes in the milky stage
(R-6.0). = LSD
T
(P = 0.05). (From: Du Plessis, 1997). (Source: African Crop Science Journal)
Based on the results of this study by Du Plessis (1997), it is proposed that insecticides be
applied when at least 20% damage per head occurs, in order to promote timely control
measures. Taking into account that the preferential feeding sites of H. armigera are not the
achenes, as well as the ability of plants to compensate for damage to florets and achenes, a
significant number of larvae can, however, be tolerated without any significant effect on
yield. Actual damage is therefore the only criterion which can be used in the determination of
economic injury levels for control of African bollworm on sunflower (Du Plessis, 1997). The
large volumes of insecticides applied for H. armigera control is therefore not justifiable, since
damage levels greater than 20% seldom occur and natural enemies are abundant.
Approximately 170 parasitoid species and a large number of predators of H. armigera have
been reported from southern and east Africa (Cherry et al., 2003). Furthermore, a nuclear
polyhedrosis virus that occurs freely in nature, contribute significantly in controlling H.
armigera populations in southern Africa.
CONCLUSION
Although many insect species occur on sunflower in Africa, only a few of these obtain
pest status. Insecticide application for control of pest species should therefore be done with
care since economic injury levels of all, except for H. armigera, have not been determined
and natural enemies are plentiful. Further research on the hemipteran pest complex of this
crop is needed since the presence of these pests may lead to qualitative crop losses.
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Insect Pests of Sunflowers in Africa 237
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas 2014 Nova Science Publishers, Inc.
Chapter 11
SOIL AMENDMENTS AND THEIR EFFECTS
ON SUNFLOWER GROWTH
Fernando Lpez-Valdez
1
*
, Fabin Fernndez-Luqueo
2
,
Perla Xchitl Hernndez-Rodrguez
1
, Minerva Rosas-Morales
1
and Silvia Luna-Surez
1
1
Centro de Investigacin en Biotecnologa Aplicada, Instituto Politcnico Nacional,
Tepetitla de Lardizbal, Tlaxcala, Mxico
2
Natural Resources and Energy Group, Cinvestav-Saltillo,
Coahuila, Mxico
ABSTRACT
An interesting topic in agriculture is the search for forms of fertilisation that have a
low impact on soil, plants, humans, and the environment. Wastewater treatment plants
almost always separate the organic matter, called wastewater sludge or sewage sludge.
This sludge is rich in mineral macronutrients such as nitrogen (ammonium or nitrate),
phosphorous (phosphate), potassium, and micronutrients. The sewage sludge might
provide the majority of necessary nutriments for growing plants, and also provide many
beneficial effects when it is applied to soils, resulting in an improvement in the chemical,
physical, and biological characteristics. We know that wastewater sludge and soil
microorganisms play an important synergetic role on the released and available nitrogen.
Although urea is the most accepted type of fertiliser used worldwide, it does have some
inconvenient drawbacks such as pH changes, microflora modifications from the soil, and
others; these issues must be considered in order to avoid N losses. An interesting
alternative might be the use of Plant Growth-Promoting Rhizobacteria (PGPR, soil
bacteria that colonize the roots of plants by inoculation onto seeds or roots and that allow
enhanced plant growth) as helpers. Examples such as Pseudomonas, Azotobacter,
Burkholderia, Klebsiella, and Bacillus, among other genera, have been reported. In this
*
Corresponding author: F. Lpez-Valdez, Centre for Research in Applied Biotechnology (CIBA) - Instituto
Politcnico Nacional. Carr. Est. Sta. Ins Tecuexcomac Tepetitla km. 1.5 s/n, Tepetitla de Lardizbal,
Tlaxcala, C.P. 90700, Mxico. Tel.: +52 55 5729 6000 ext. 87805; Fax: +52 248 487 0762. E-mail address:
flopez2072@yahoo.com.
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 240
chapter, we focus on specific types of fertilisation or soil amendments and their effects on
this important crop. In particular, Bacillus subtilis has showed promising results. It is
known that these bacteria might improve plant growth by several direct mechanisms as
well as indirect mechanisms, such as controlling phytopathogen organisms, or a
combination of both. A regular strain of Bacillus subtilis was co-inoculated with urea in
sunflower roots, and it was found that the strain temporarily stimulated sunflower cultivar
growth. We found that B. subtilis improves the sunflower establishment, particularly its
strength and vigour during the early stages, resulting in an increase of the mass and
length of roots compared with the control treatment. Application of either organic or
mineral fertiliser improved the crop yield under various conditions, but fertilising the
crops with sewage sludge might be more environmentally friendly than using mineral
fertiliser. Wastewater sludge has a high NH
4
+
content that is slowly oxidized to NO
3
-
while being absorbed by sunflower cultivars. In both cases (organic amendment or
bacterial inoculation with urea), the NO
3
-
is not lixiviated or leaked during experiments.
Accordingly, the wastewater sludge and B. subtilis might be potential methods of
fertilisation for sunflowers or any ornamental cultivars.
Keywords: Bacillus subtilis, fertilisers, sunflower, urea, wastewater sludge
INTRODUCTION
Agriculture is an essential discipline that concerns the possible cultivation of domestic
plants in order to improve food or biomass productions. Enhancing food security and a
sustainable production of foods (agricultural sustainability) are modern global topics.
Therefore, fertilisation is a critical topic, particularly aspects such as the reasonable
application of fertilisers, reutilization of materials (wastewater sludge, manure, tannery
sludge, whey, biochar, inter alia), and application of beneficial microorganisms (PGPRs,
mycorrhizal fungi, among other microorganisms) as soil amendments. These could all be
strategies that lead to a sustainable agriculture. In this chapter, we explore some of the
strategies that we have used to experiment on an important crop, the sunflower.
SUNFLOWER
The sunflower (Helianthus annuus L.) is a significant crop and an attractive ornamental
plant known throughout the world. This crop is a considerable source of edible oil, protein
(Nesterenko et al., 2013; Lin et al., 1974), oil for diesel biosynthesis, phytoextraction, and
even animal feed. It shows a well-developed and deeply penetrating root system, allowing a
good establishment, which is why it is considered a drought-tolerant plant (Stone et al.,
2002). Therefore, the sunflower is easily cultivated, exhibiting growth with minimal or no
fertilisation (Ruiz and Maddonni, 2006), and even exhibiting growth under rain-fed
conditions. Mexico might be the origin of domestication of this crop. There is evidence of
some early remains of H. annuus discovered at the San Andrs site in the Gulf Coast region
of Tabasco, Mexico, constituting the earliest record of the domesticated sunflower (Lentz et
al., 2001; Wills and Burke, 2006).
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Soil Amendments and Their Effects on Sunflower Growth 241
CHEMICAL FERTILISERS
It is known that nitrogen (N) is the most important mineral nutrient for non-leguminous
plants. This mineral has several chemical forms within the soil, such as ammonium (NH
4
+
-N),
nitrite (NO
2
-
-N), and nitrate (NO
3
-
-N); ammonium and nitrate are both valuable ions for
plants. There are several synthetic N fertilisers, such as ammonia, urea, ammonium nitrate,
calcium ammonium nitrate, ammonium sulphate, and urea ammonium nitrate, among others.
The world's production of ammonia (NH
3
) exceeded 134.2 x 10
9
kg y
-1
in 2011 (IFA, 2013).
With the highest N content (82%), it is feedstock for N fertilisers and other products.
However, when ammonia is applied as aqua ammonia (dissolving ammonia in water from
20% to 24% N solution) as fertiliser, special care must be taken: it must not be placed in close
proximity to seeds, and it must be handled carefully for safety purposes.
Urea is the most common N fertiliser used worldwide, constituting over 71.1 x 10
9
kg y
-1
in 2011 (IFA, 2013). The production of ammonia and urea is increasing annually; in fact, the
ratio of world urea production is about 2.1 x 10
6
kg y
-1
(the result was estimated from data
from 2002 to 2011). Urea has several advantages over others fertilisers, such as a high N
content (46.7%) and solubility (20 C) at 1,080 g L
-1
. In addition, it is easier to handle than
NH
4
NO
3
or ammonia, easier to store, less corrosive to machinery, and less likely to explode
or burn. However, some problems have been reported, including damage to seeds, seedlings
or young plants; NO
2
-
toxicity; phytotoxicity of foliar-applied urea; and volatilization of urea-
N as NH
3
(Bremner, 1995).
WASTEWATER SLUDGE
Wastewater sludge or sewage sludge is an unavoidable by-product from wastewater
treatment plants. The wastewater sludge is rich in organic matter as well as macro- and
micronutrients for plants (N, P, S, and minerals). It can even provide C and energy sources for
the growth of soil microorganisms (Lpez-Valdez et al., 2010, 2011b). Also, it has the
potential to improve the physical, chemical, and biological properties of the soil. However,
sewage sludge can also contain some pathogensbacteria, virus, protozoa cysts, or helminth
ova (Jimnez-Cisneros et al., 2001; Jimnez, 2007)which are heavy metals and organic
pollutants that must be eliminated. According to USEPA, sewage sludge can be classified as
Class A or Class B with regard to pathogens (2013). Class A refers to sludge that might be
amended to agricultural soils with edible cultivars. In contrast, Class B refers to sewage
sludge that might to applied to non-agricultural soils or even soils where ornamental plants
will be cultivated.
Approximately 37% of the sewage sludge produced in the world was used in agriculture
and 12% was used in forestry (Fytili and Zabaniotou, 2008). The remainder of the sludge was
not used, commonly resulting in its being confined or burned. Application of wastewater
sludge in agriculture is an alternative disposal approach that, relative to the burn-up approach,
offers the opportunity to recycle nutrients that enhance plant growth, return organic matter to
the soil (Lpez-Valdez et al., 2011b), and avoid CO
2
-C return to the atmosphere. It has been
reported that sludge significantly increases the yield in apple trees (Malus pumila Mill.)
(Bozkurt et al., 2010), commun beans (Phaseolus vulgaris L.) (Fernndez-Luqueo et al.,
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 242
2010), cotton (Gossypium hirsutum L.) (Samaras et al., 2008), mung beans (Vigna radiata L.
cv. Malviya janpriya) (Singh and Agrawal, 2010b), rice (Oryza sativa L. cv. Pusa sugandha)
(Singh and Agrawal, 2010a), soybeans (Glycine max (L.) Merrill) (Souza et al., 2009), and
sunflowers (Helianthus annuus L.) (Lavado, 2006).
The goal of sewage sludge application into the soil is to provide an alternative, to restore
degraded or N-depleted soils and simultaneously enhance plant growth (Fernndez-Luqueo
et al., 2009; Lpez-Valdez et al., 2010, 2011b).
BACTERIA AS HELPERS (PGPR)
Another compelling alternative is the application of microorganisms such as bacteria and
filamentous fungi (as mycorrhizal fungi). Bacteria are the most abundant microorganisms in
soil (up to 6 x 10
8
cells g
-1
of soil and a weight of approximately 10,000 kg ha
-1
) (Kilian et al.,
2000). The number of bacteria found in soil depends on the season, the type of soil, the
cultivar used, the moisture content, the oxygen supply in the soil, the tillage and fertilisation
of the soil, the penetration of the soil by plant roots, and the depth from which the soil
samples were taken (Kilian et al., 2000). Pseudomonas, Arthrobacter, Achromobacter,
Clostridium, Micrococcus, Flavobacterium, Azospirillum, Azotobacter, and Bacillus are the
most representative genera of bacteria from soil and other environments (Lpez-Valdez et al.,
2011a). Bacteria have notorious advantages over fungi such as faster growth, a ubiquitous
presence, and the ability to be cultured in vitro. Bacteria might even compete for an
ecological niche; bacteria produces metabolites or enzymes that enhance or stimulate the
growth of plants through direct or indirect mechanisms. The Bacillus genus shows a wide
spectrum of mechanisms that might (a) stimulate plant growth as fungistatic or bactericidal
compounds (Singh and Deverall, 1984; Ongena et al., 2005; Forchetti et al., 2007; Sang et al.,
2008; Swain and Ray, 2009; Alvindia, 2013), (b) produce phytohormones involved in root or
shoot growth (Arajo et al., 2005; Yao et al., 2006; Karadeniz et al., 2006; Forchetti et al.,
2007; Swain and Ray, 2009; Ahmed and Hasnain, 2010), (c) induce systemic resistance
(Gupta et al., 2000) by volatile organic compounds (Ryu et al., 2003; Ping and Boland, 2004),
(d) colonize plant roots (Dijkstra et al., 1987) by remaining very close to the root tip by
passive displacement, and (e) produce extracellular enzymes. Another interesting result is the
reduction of ethylene production, as described by Penrose and Glick (2003). Also, the
Bacillus genus is known to affect the fruit ripening, leaf senescence, flower abscission,
germination, cell elongation and proliferation, nodulation, and response to plant pathogen
attack. Hence, bacteria that can stimulate or promote plant growth are called PGPR.
SAMPLING SITE AND PROPERTIES OF SOIL
We used several soils from different land uses. The first sampling site was located in the
former lake of Texcoco (Mexican valley, Mexico; 1930 N, 9853 W) at an altitude of
2,250 m above sea level. This soil is alkaline saline, classified as Typic Ustifluvent with a pH
between 8.5 and 11, electrolytic conductivity (EC) between 4 and 170 dS m
-1
, and water
holding capacity (WHC) of 684 g kg
-1
soil. The former lake bed is not cultivated, but some
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Soil Amendments and Their Effects on Sunflower Growth 243
grasses and small trees can grow. More details are available in Lpez-Valdez et al. (2010).
The second soil samples were obtained from Otumba (Estado de Mxico, Mexico; 1942 N,
9849 W), near the former lake Texcoco. The soil, classified as Typic Fragiudepts, was
characterized as sandy loam, pH 7.6, EC of 1.1 dS m
-1
, WHC of 530 g kg
-1
soil, and an
organic C content of 7.2 g C kg
-1
soil. This soil was cultivated with maize, receiving a
minimum amount of mineral fertiliser without being irrigated. More details are available in
Fernndez-Luqueo et al. (2009). The third sampling site is located in Alcholoya, an Acatln
village (Hidalgo, Mexico; 2,120 m above sea level and 2009 N, 9826 W). The soil was
classified as Typic Fragiudepts with pH 6.5, EC 0.7 dS m
1
, a WHC of 846 g kg
1
soil, an
organic C content of 11.1 g kg
1
soil, and a total N content of 1.0 g kg
1
soil. This soil
received organic fertiliser (cow excreta) occasionally. More information is available in
Lpez-Valdez et al. (2011b). All soils were sampled by augering (0 to 15 cm depth) from
three plots equivalent to 0.5 ha. Soils from each plot were pooled and sieved. In total, three
soil samples were obtained.
CHARACTERISTICS OF THE WASTEWATER SLUDGE
Reciclagua (Sistema Ecolgico de Regeneracin de Aguas Residuales Ind., S.A. de C.V.)
in Lerma (Estado de Mxico, Mexico) treats wastewater from various sources, mainly
alimentary industries and households. In the primary treatment, the wastewater is mixed with
a flocculant, and the sludge obtained is passed through a belt filter in order to reduce the
water content. Although the concentration of heavy metals and toxic organic compounds has
been low, (Franco-Hernndez et al., 2003) this sludge can be classified as Class B due to its
pathogen content. The characteristics of sludge were pH 8.1, CE 7.9 dS m
-1
, water content
847 g kg
1
, organic C content 288 g kg
1
, and total N content 41.8 g kg
1
. The mineral N was
NH
4
+
-N 13 g kg
-1
, NO
2
-
-N 8.3 mg kg
-1
, and NO
3
-
-N 122 mg kg
-1
on a dry matter.
These characteristics show that sewage sludge is a strong candidate for an organic
fertiliser, since the C/N ratio is over 14, meaning that sludge has a high N content. Almost
30% is ammonium, indicating that the mineral N is immediately available for plants; the
remaining 70% is organic N (such as proteins, amino acids, and nucleic acids, among others),
available for microorganisms that mineralize organic N up to mineral nitrogen. This mineral
N is released slowly and remains available for plants.
BACI LLUS SUBTI LI S
We studied a regular strain of Bacillus subtilis that was isolated from the potato
rhizosphere (identified by 16S ribosomal RNA, rRNA, gene sequencing). The strain was
provided by Dr. Olalde-Portugal (Laboratory of Microbial Ecology, Department of
Biotechnology and Biochemistry, Cinvestav, Gto., Mexico). This strain was tested and
selected due to its ability to provide positive results in some in vitro tests, thus demonstrating
its antagonistic activity against some phytopathogenic fungi (Fusarium oxysporum and
Rhizoctonia solani AG1). Also, this strain embodies many characteristics, such as the ability
to solubilize phosphate as described by Burr et al. (1984), the 1-aminocyclopropane-1-
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 244
carboxilate deaminase enzymatic activity as reported in Penrose and Glick (2003), the indole-
3-acetic acid production by the colorimetric method (Azcn et al., 2009), and the root
colonization on maize and sunflower in a Petri dish using Phytagel (Sigma Co.) as a medium
(Dijkstra et al., 1987). This strain belongs to a collection that is being explored as PGPR. In
our opinion, this is a regular or common strain. More information can be found in Lpez-
Valdez et al. (2011a).
METHODS
The sunflower seeds were provided by the Departamento de Fitotecnia, Universidad
Autnoma de Chapingo, Texcoco, Estado de Mxico, Mexico [Department of Plant Science,
Autonomous University of Chapingo, Texcoco, Estado de Mxico, Mexico]. The non-treated
seeds were received for amendments with sewage sludge, urea, or unamended soil. However,
according to Lpez-Valdez et al. (2011a), the seeds were disinfected for inoculation with B.
subtilis and were incubated in an agar-agar plate under aseptic conditions in order to
determine whether microorganisms grow after one day of incubation. Afterward, these seeds
were dressed with a suspension of B. subtilis at 10
7
CFU mL
-1
in 1% carboxymethylcellulose.
One hundred and eight sub-samples were prepared as follows: each sub-sample contained
6.5 kg soil in a cylindrical pot with the purpose of obtaining five treatments, three plots, three
replicates, and three sampling times (at 37, 60, and 95 days after sowing) during the
experiment (Lpez-Valdez et al., 2011b). At the onset of the experiment, 0.5 g urea was
added to nine pots from each of the three sampled plots; twelve days after emergence, the
nine plantlets were amended with another 0.5 g urea (equivalent to 150 kg N ha
-1
, the UREA
treatment). Nine others pots were amended with 30 g sludge (150 kg N ha
-1
, the SLUDGE
treatment), nine were left unamended (the PLANT treatment), and nine were left unamended
and unsown (the CONTROL treatment). The last treatment was amended with B. subtilis (the
BACTERIA treatment); the seeds were disinfected, dried, and dressed with the B. subtilis
suspension (as described above). In this treatment, the plants were fertilised with 0.5 g urea
(75 kg N ha
-1
). Each pot was sown with three sunflower seeds. Eight days after emergence,
two of the three plantlets in each pot were discarded. The tap water was analysed, 19 kg
mineral-N ha
-1
was additionally added to each treatment during the whole experiment, and no
water was leached out from the pots (Lpez-Valdez et al., 2011b).
At the onset of the experiment, approximately every two days up to 30 days after sowing,
the columns were airtight and the atmosphere was analysed for CO
2
and N
2
O at 0, 3, 15, and
30 minute intervals. On every sample day (37, 60, and 95 days), nine pots were selected at
random from each treatment. Soil samples were collected at depths of 0-15 cm and 16-30 cm.
The roots were separated from the shoots. The variables measured in the plants were shoot
height, root length, fresh shoot weight, fresh root weight, dry shoot weight, dry root weight,
seed weight per plant, number of seeds per plant, and total N content; in the soil, the variables
measured were pH, EC, NH
4
+
-N, NO
2
-
-N, and NO
3
-
-N. Also, the gas production was
measured as CO
2
-C and N
2
O-N. Further details can be found in Lpez-Valdez et al. (2011b).
Significant differences between plant and soil characteristics as a result of the various
treatments were determined by analysis of variance (ANOVA) and based on the least
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Soil Amendments and Their Effects on Sunflower Growth 245
significant difference test, using the general linear model procedure (PROC GLM; SAS
Institute Inc., 1989).
UREA NOTES
Sunflower Characteristics (Alcholoya Soil)
According to our results, the UREA treatment was not significantly different in shoot
height and dry root weight compared with the other treatments at one, two, and three months.
After two months, the root length and fresh root weight were similar to the BACTERIA and
PLANT treatments. Finally, the effect of urea was significantly different in the dry shoot
weight compared with the PLANT treatment. Compared with the unfertilised plants, the urea
enhanced the dry shoot weight, causing a significant increase of total N content per kg of
dried plant (P<0.05). As we know, the urea has a disadvantage: it decomposes up to NO
3
-
,
which could form leaching, and it continues decomposing and losing N
2
O through the
nitrification or denitrification processes.
Soil Characteristics of Alcholoya
The pH did not significantly differ by treatment in either soil depth (0-15 and 16-30 cm).
At the end of the experiments, EC was not significantly different at a depth of 0-15 cm, but a
slight increase (not a significant difference) was noted at a depth of 16-30 cm. Fernndez-
Luqueo et al. (2009) showed that soil amended with urea and cultivated with common bean
did not result in a change of pH. Mineral N, NO
2
-
-N, and NH
4
+
-N did not significantly differ
by treatment in either soil depth. However, the treatments with urea (the UREA and
BACTERIA treatments) showed significant NO
3
-
-N differences compared with the
unfertilised plants. The urea-N was transformed to NO
3
-
-N almost totally, and remained in the
soil at the given depth (16-30 cm). As reported by Casado-Vela et al. (2006), tap water
provides mineral N at the end of the experiments. An analysis by principal components
(PCA) showed that the PLANT treatment is opposite to the UREA and BACTERIA
treatments (quadrant +,+) in the PCA plot (Figure 1); these results indicate that the treatments
have positive effects on sunflower plants, in contrast to sunflower plants without some kind
of fertilisation. The UREA and BACTERIA treatments have effects on the variables
measured on soil, i.e., increasing in naturally the N
2
O and CO
2
productions, EC, and NO
3
-
(in
both depths) as consequent of urea application and its decomposition (to CO
2
and NH
3
). Also
minor effects have on pH, NH
4
+
, NO
2
-
due to nitrification process (where NH
4
+
was
transformed to NO
2
-
and NO
2
-
was transformed to NO
3
-
). The urea addition had little effect on
the pH of the soil in these experiments, but is well known that long-term applications of urea
decrease the pH of soil. In the long-term, ammonium decreases soil pH by oxidation to NO
3
-
,
generating a proton (Enwall et al., 2007). The sunflowers fertilised with urea did not affect
the production of CO
2
; however, the production rate of N
2
O was more than double that of the
mean produced by unamended soil.
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 246
WASTEWATER SLUDGE NOTES
Otumba and Texcoco Soils
When sludge or sterile sludge was applied to an alkaline saline soil (Texcoco) or a
depleted nitrogen soil (agricultural soil from Otumba), dynamic profiles of CO
2
-C in both
soils showed that the microorganisms of sludge were affected by the alkalinity and salinity of
Texcoco soil as opposed to agricultural soil. On the other hand, the ammonium dynamics
showed that microorganisms immobilise the ammonium from several treatments of sludge,
sterile sludge, or ammonium sulphate, on the third day of the experiment. After that, the
ammonium was released, and later, the ammonium concentration was consumed. The NH
4
+
-N
dynamics showed that soil microorganisms immobilise N, release it, and finally, transform it
to NO
3
N; in other words, the N became available for assimilation by the plants. The final
concentration of NO
3
-
-N was higher in amended soil with sludge and sterile sludge than with
ammonium sulphate (Lpez-Valdez et al., 2010). Both soils were considered as nitrogen
deficient: NH
4
+
-N concentration was 10 mg N kg
-1
dry soil in both soils, and NO
3
-
-N
concentration was 30 and 10 mg N kg
-1
dry soil for Otumba and Texcoco soils, respectively.
These results suggest that wastewater sludge could be a useful organic fertiliser. Nevertheless,
the pathogenic microorganisms in the sludge must be reduced.
Sunflower Characteristics (Alcholoya Soil)
The amended soil with sludge showed no significant difference in shoot height compared
with the other treatments at any sampled time. There was a significant difference between the
fresh and dry shoot weights of the PLANT and UREA treatments during the first two months.
Fresh and dry root weights were similar to the UREA treatment, but they were significantly
different to the PLANT treatment (P<0.05). The sludge was not different in the number of
seeds per plant and total N content mean compared with the UREA and PLANT treatments,
but the seed weight per plant was significantly different in the SLUDGE treatment. It has
been reported (Singh and Agrawal, 2010b) that sludge could increase the shoot length in
crops such as wheat (Triticum sp.), mung bean (Vigna radiate L. cv. Malviya janpriya), and
kenaf (Hibiscus cannabinus L.), but in this work it was not observed. One explanation could
be that the three experiments were begun at different seasons; it is well known that the
weather and amount of sunlight have strong effects on the growth of crops. Christodoulakis
and Margaris (1996) reported that sewage sludge amendment improved the maize growth in
comparison to commercial fertiliser, increasing plant height by 77%, whereas commercial
fertiliser increased plant height by 25%, compared with the control treatment. Liu et al.
(2009) reported an incremental increase of plant height from soil amended with 10% sludge
(doses from 0% to 30% sludge); additionally, they found that fresh weight, chlorophyll
content, activities of peroxides (POD) and catalase (CAT), or decreasing contents of
malondialdehyde (MDA) and membrane permeability (MP) increased the sewage sludge ratio
from 0% to 10% sludge. This sludge had 2% N kg
-1
, and it was disinfected by chlorination.
The PCA revealed that the SLUDGE treatment could actually surpass the UREA and PLANT
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Soil Amendments and Their Effects on Sunflower Growth 247
treatments because sludge has a positive effect on the total N content of plants as well as the
weight and height of shoots (Figure 2).
Soil Characteristics of Alcholoya
At the end of the experiment, the EC was similar to the UREA and PLANT treatments in
both depths, but the SLUDGE treatment was significantly different in soil pH, reaching a
final value of 7.4 and 7.1 in depths of 0-15 cm and 16-30 cm, respectively. The soil pH of
Alcholoya had an average of 6.5. Additionally, at the end of the experiments, the NO
2
-
concentration was significantly different in the SLUDGE treatment at a depth of 0-15 cm, but
it was not different at a depth of 16-30 cm. Final concentrations ranged from 0.33 to 0.43 mg
NO
2
-
-N kg
-1
dry soil for a depth of 0-15 cm, and 0.23 to 0.26 mg NO
2
-
-N kg
-1
dry soil in a
depth of 16-30 cm. The concentrations were low because this ion is an intermediate for NO
3
-
production. Interestingly, the dynamic of the NO
3
-
concentration resulted in a higher
concentration during the first month and a lower concentration at the end of the experiments
in both depths; in addition, non-leaching was found. Furthermore, we found that the NO
3
-
concentration was significantly different for the UREA treatment compared with the other
treatments in both soil depths (P<0.05) (Table 1).
The lowest residual concentration of NO
3
-
-N was 35%, found in the SLUDGE treatment
at a depth of 16-30 cm. These results could suggest that sludge decomposes slowly. The NO
3
-
-N concentration was similar to unfertilised plants.
Figure 1. Principal components analysis of unfertilised and fertilised sunflower plants. The PLANT
treatment refers to unfertilised sunflowers, the UREA treatment refers to fertilised sunflowers with 1 g
urea (150 kg N ha
-1
), and the BACTERIA treatment refers to sunflower seeds inoculated with B. subtilis
and fertilised with urea at 75 kg N ha
-1
. The experiments were carried out under greenhouse conditions.
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 248
Figure 2. Principal components analysis of unfertilised and fertilised sunflower plants. The PLANT
treatment refers to unfertilised sunflowers, the UREA treatment refers to fertilised sunflowers with 1 g
urea (150 kg N ha
-1
), and the SLUDGE treatment refers to amended sunflowers with sewage sludge at
150 kg N ha
-1
.
The dynamic of ammonium concentration at a depth of 0-15 cm was significantly higher
in the PLANT treatment (unfertilised plants) and was significantly different with regard to the
SLUDGE treatment, but it was not different with regard to the UREA treatment. This increase
in ammonium concentration could be due to an inlet of N from tap water; there was no N
supply from another source. On the soil depth of 16-30 cm, the dynamic was similar, but the
NH
4
+
-N concentration showed a significant decrease in the SLUDGE treatment: from 8.0 to
6.4 mg N kg
-1
soil (P<0.05) (Table 2).
Table 1. Dynamics of NO
3
-
-N (mg N kg
-1
soil) concentration by treatment in both
Alcholoya soil depths. The PLANT treatment refers to unfertilised sunflowers,
the UREA treatment refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
,
and the SLUDGE treatment refers to amended sunflowers with sewage sludge
at 150 kg N ha
-1
. The experiments were carried on under greenhouse conditions
Months PLANT UREA SLUDGE
0 15 cm depth
1
st
11.8 B
a
c
b
33.1 A a 22.4 A b
2
nd
10.7 B c 36.1 A a 22.5 A b
3
rd
14.9 A b 19.4 B a 14.6 A b
16 30 cm depth
1
st
17.0 A c 49.8 A a 34.0 A b
2
nd
12.7 B b 35.0 B a 18.4 B b
3
rd
11.7 B b 29.3 B a 12.2 B b
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not significantly different over time.
b
Values with the same letter are not significantly different between treatments.
SLUDGE
PLANT
UREA
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
PC2: 28%
PC1: 41%
Shoot dry weight
Root fresh weight
Number of seeds
Seed weight
Shoot length
Root dry weight
Root length
Shoot fresh weight
Total N content
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Soil Amendments and Their Effects on Sunflower Growth 249
Table 2. Dynamics of NH
4
+
-N (mg N kg
-1
soil) concentration by treatment from
Alcholoya soils. The PLANT treatment refers to unfertilised sunflowers, the UREA
treatment refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
,
and the SLUDGE treatment refers to amended sunflowers with sewage sludge
at 150 kg N ha
-1
. The experiments were carried on under greenhouse conditions
Months PLANT UREA SLUDGE
0 15 cm depth
1
st
7.0 B
a
b
b
9.2 A a 8.8 A ab
2
nd
4.3 B b 14.1 A a 6.2 B b
3
rd
11.7 A a 9.6 A ab 8.1 A b
16 30 cm depth
1
st
6.7 B b 8.7 A a 8.0 A a
2
nd
4.0 B b 6.2 A a 4.9 C ab
3
rd
10.4 A a 8.9 A ab 6.4 B b
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not significantly different over time.
b
Values with the same letter are not significantly different between treatments.
In the SLUDGE treatment, the rate of CO
2
production increased to 86% (1.4 mg C kg
-1
day
-1
) with regard to the uncultivated and unamended soil (CONTROL treatment). The
SLUDGE treatment had a significant difference in the rate of N
2
O production compared with
all other treatments: the mean production was 2.31 g N kg
-1
day
-1
, which was 12 times that
of the CONTROL treatment. The wastewater sludge refers to organic mattermatter that
involves a high production of greenhouse gasses by decomposing microorganismsbut there
is no such comparison when sludge is burned. The sewage sludge used in these experiments
had an organic C content of 28.8%, meaning about 18,000 mg CO
2
-C kg
-1
dry sludge (on a
dry matter basis). PCA showed that the SLUDGE and UREA treatments had a major effect
over all soil parameters. The SLUDGE treatment affected the ammonium and nitrate in both
soil depths and the EC at a depth of 0-15 cm; the UREA treatment affected the pH in both soil
depths, principally (Figure 3).
Recent Work
Recently, we have studied wastewater sludge in order to remove or reduce the amount of
pathogen microorganisms such as Salmonella sp., faecal coliforms, coliphage, protozoa cysts,
and helminth ova. The sewage sludge was characterised in terms of the initial concentrations
of helminth ova that were determined. Viability was determined by continuous washing,
combined with several filtration steps: suspension, concentration, and incubation in H
2
SO
4
0.1 N at 26 C for 30 days (USEPA, 2013). One kg of wastewater sludge was placed in
plastic containers of 0.30 0.25 0.12 m dimensions, and 100 g CaO was added. The sludge
was exposed by 15-day increments from day 0. Viable ova quantification was performed at 0,
5, and 15 days after the start of treatment.
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F. Lpez-Valdez, F. Fernndez-Luqueo, P. X. Hernndez-Rodrguez et al. 250
Figure 3. Principal components analysis of unfertilised and fertilised sunflower plants and their effects
on soil properties. The PLANT treatment refers to unfertilised sunflowers, the UREA treatment refers
to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the SLUDGE treatment refers to amended
sunflowers with sewage sludge at 150 kg N ha
-1
.
Table 3. Viable ova count by treatment and by sampling day (g
-1
total solids).
The experiments were carried out under two different conditions
(cold and warm seasons)
Day
Alkaline Non-Alkaline
MSD
c
Ova g
-1
TS
0 3053 A
a
a
b
3470 A a 2077
5 1387 A a 1805 A b 2960
15 0 B a 1943 A b 773
MSD 3110 1207
a
Values with the same capital letter are not significantly different between treatments.
b
Values with the same letter are not significantly different over time.
c
MSD: Minimal significant difference (P<0.05).
We found that the lime used significantly decreased embryonic development of helminth
ova from Day 15, when we did not find viable ova. The alkaline treatment increased the
temperature from 15C to 33C on the first day. Furthermore, the lime dose increased the pH
of the sewage sludge to 12 units upon contact; the pH remained above this value for 72 hours
(Table 3). Finally, treatment with the lime at 10% allowed for the inactivation of helminth
ova. However, future studies should be conducted in order to confirm the inactivation of
helminth ova in limed sewage sludge treated under large scale or field conditions. We are
interested in researching other treatments that reduce pathogen microorganisms, in order to
reach a Class A sewage sludge that can amend agricultural soils.
SLUDGE
PLANT
UREA
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
PC2: 17%
PC1: 22%
pH, 15-30 cm
pH, 0-15 cm
EC, 0-15 cm
NO
2
-
,
0-15 cm
Production of CO
2
EC, 15-30 cm
NO
2
-
, 15-30 cm
NO
3
-
, 0-15 cm
NO
3
-
, 15-30 cm
NH
4
+
, 0-15 cm
NH
4
+
, 15-30 cm
Production of N
2
O
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Soil Amendments and Their Effects on Sunflower Growth 251
BACI LLUS SUBTI LI S NOTES
Sunflower Characteristics (Alcholoya Soil)
The shoot height and dry root weight characteristics were not significantly different
between treatments (P<0.05). However, at the end of the experiment, the fresh and dry shoot
weight characteristics were significantly different compared with the UREA and PLANT
treatments. The root length and fresh root weight parameters showed a significant difference
with regard to the UREA and PLANT treatments at the start of the experiment, but not at the
end. Meanwhile, parameters such as weight of seeds, number of seeds, and total N content of
plants did not differ significantly throughout the entire experiment (P<0.05). As mentioned
above, this bacterium is a regular microorganism. As such, the tested strain produces IAA,
solubilizes phosphorus, and tests positive for ACC deaminase, but it was not an outstanding
PGPR. The application of B. subtilis to plants cultivated in soil amended with urea increased
root growthnamely, the root length and root weightas opposed to plants only fertilised
with urea at the first month. We found that the B. subtilis strain had a temporary stimulation
effect on the sunflower crop at the first month; similar results were reported by Swain and
Ray (2009).
Figure 4. Principal components analysis of unfertilised and fertilised sunflower plants and their effects
on soil properties. The PLANT treatment refers to unfertilised sunflowers, the UREA treatment refers
to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the BACTERIA treatment refers to
sunflower seeds inoculated with B. subtilis and fertilised with urea at 75 kg N ha
-1
.
BACTERIA
PLANT
UREA
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1.25 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25
PC2: 21%
PC1: 33%
pH, 15-30 cm
pH, 0-15 cm
EC, 0-15 cm
NO
2
-
,
0-15 cm
Emission of CO
2
EC, 15-30 cm
NO
2
-
,
15-30 cm
NO
3
-
, 0-15 cm
NO
3
-
, 15-30 cm
NH
4
+
,
0-15 cm
NH
4
+
,
15-30 cm
Emission of N
2
O
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The PCA showed that the BACTERIA treatment had a positive effect on the length and
weight of shoots; meanwhile, the UREA treatment had a positive effect on the length and
weight of roots (Figure 4). In contrast, the unfertilised plants had negative PC1 and PC2,
meaning less development of roots and shoots as well as fewer and lighter seeds. On the other
hand, the BACTERIA treatment was established with half of the N amount with regard to the
UREA treatment. Also, unfertilised plants showed chlorotic leaves with regard to the UREA
and BACTERIA treatments.
Soil Characteristics of Alcholoya
Treatments had no effect on the pH in either soil depth. However, the soil pH increased
significantly at the end of the experiments. In the case of EC, the BACTERIA, UREA, and
PLANT treatments showed no significant difference at a depth of 0-15 cm by the end of the
experiment (P<0.05). For the soil depth of 16-30 cm, the first two months were significantly
higher for the UREA and BACTERIA treatments, but by the end of the experiment, the
BACTERIA treatment showed a significant difference with regard to unamended plants. Urea
application seem to affect the EC. Otherwise, these increments could be due to the extra
minerals supplied by tap water (high contents of Ca
+2
, Mg
+2
, and Na
+
) that were increasing
over time (Lavado, 2006). The NO
2
-
concentration was similar between treatments in both
soil depths throughout the entire experiment. Regarding NO
3
-
-N, we found no significant
difference between the UREA and BACTERIA treatments, but we did find a difference with
regard to unamended plants (P<0.05). Also, the analysis shows that the NO
3
-
concentration
decreased significantly over time (Table 4).
Table 4. NO
3
-
-N concentration by treatment in both Alcholoya soil depths.
The PLANT treatment refers to unfertilised sunflowers, the UREA treatment
refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the BACTERIA
treatment refers to sunflowers inoculated with B. subtillis plus urea at 75 kg N ha
-1
NO
3
-
-N (mg N kg
-1
soil)
Months PLANT UREA BACTERIA
0 15 cm depth
1
st
11.8 B
a
c
b
33.1 A a 43.1 A a
2
nd
10.7 B c 36.1 A a 27.2 B a
3
rd
14.9 A b 19.4 B a 19.8 B a
16 30 cm depth
1
st
17.0 A c 49.9 A a 49.9 A a
2
nd
12.7 B b 35.0 B a 28.7 B a
3
rd
11.7 B b 29.3 B a 32.3 B a
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not significantly different over time.
b
Values with the same letter are not significantly different between treatments.
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Soil Amendments and Their Effects on Sunflower Growth 253
All treatments were significantly similar for the ammonium concentration in both soil
depths at the end of the experiment. Cultivating the sunflower did not affect the CO
2
production in comparison with the uncultivated soil. Sunflower roots inoculated with B.
subtilis plus urea had no effect on CO
2
production compared with urea-amended soil. It is
known that cultivated soil increases the CO
2
production due to root exudates and roots and
leaves dying; these are easily decomposable organic material degraded by the soil and
rhizospheric microorganisms. On the other hand, the addition of urea to the soil doubled the
mean N
2
O production rate compared with the unamended soil. The N
2
O production from the
soil is the result of oxidation of NH
4
+
to NO
3