**2. Sunflower breeding for resistance to biotic stresses**

Concerning biotic stresses in sunflowers, it can be safely concluded that diseases caused by different fungi present the most serious problem. Broomrape, the parasitic angiosperm, is in the second place, viruses and bacteria in third and fourth [22].

#### **2.1. Sunflower diseases**

expression studies using northern blots, western blots, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), functional analyses using plant transfor‐

Among their findings was the conclusion that transcription factors are proteins able to recognize and bind specific DNA sequences present in the regulatory regions of their target genes. Upon binding, entire signalization cascades are induced or repressed and the plant can

The most amazing results obtained during these studies and other current studies are related to the divergence in structure and function of TFs and miRNAs found in sunflower, apparently conserved in some cases in other *Asteraceae* species but not in model plants. The release of the genomic sequence together with the advance in transformation techniques will certainly help to better understand how sunflower evolved to be adapted to abiotic stress factors and which

Alberdi *et al.* [46] studied the relationship between a set of molecular markers (amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR)) and leaf expansion parameters under water-deficit conditions in a cross of two public sunflower lines of contrast‐ ing response, in its F2 and F2:3 progenies, and in an independent F8 recombinant inbred line

Based on phenotypic trials (two in growth chambers – F3 and F2–3) and experiments in a greenhouse (RIL population), certain leaves collected during these experiments were used for DNA extraction. Using a set of 60 SSR and 41 AFLP markers, they achieved significant results, which may be useful for the development of molecular markers for assisted selection in breeding programs oriented to generate new cultivars with improved adaptation to water

Liu and Jan [47] closely studied the results of molecular studies about abiotic stresses in light of their own as well as other authors' research. They concluded that approaches using molecular biology, functional genomics, transcriptome, and proteomics have been used to identify genes or quantitative trait loci (QTLs) and proteins correlated with the network of the response to such stresses, which will provide knowledge for the development of hybrids with resistance or tolerance to them. Some wild species grow in locally extreme environments

Studying the phenomenon of salt tolerance in sunflower, Lexer *et al.* [48] identified an EST that codes for the Ca-dependent protein kinase with maps to a salt-tolerance QTL in sunflower.

Due to the basic structure of its main organs (root, stem, and leaves), sunflower is more resistant to abiotic stresses than other field crops. Therefore, it is usually grown on soils of lower quality ("marginal soils") and in semiarid and arid conditions, where it is often exposed to abiotic

adapt itself, at least temporarily, to the adverse conditions to which it is subjected.

Based on the copious results, Arce *et al.* [45] made the following conclusions.

novel regulating molecules are playing key roles in such an adaptation.

providing an opportunity to study species from these habitats.

(RIL) population.

stress conditions.

**1.8. Conclusions**

stresses.

mation, both stable and transient, confocal microscopy, and microarrays.

598 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

The original variability of the cultivated sunflower is very narrow and different in genes applicable in selection for the improvement of different agronomic traits, especially those conferring resistance to diseases.

Diseases are a limiting factor in the production of sunflower in all continents where it is grown. Different diseases are dominant in different growing regions, depending on the prevailing environmental conditions. Some diseases cause economic damage to sunflower in all sun‐ flower-growing regions of the world. More than 30 different pathogens that attack sunflowers and cause economic loss in production have been identified so far (Table 1). Sunflower breeders have achieved significant results in finding genes for resistance or high tolerance to certain diseases in wild species and incorporating them into cultivated sunflower genotypes possess‐ ing high combining ability [22].


**Table 1.** The most common sunflower diseases

Wild sunflower species have been a valuable source of resistance genes for many of the common pathogens of the cultivated sunflower. The relative severity of individual diseases varies widely, depending on climate and host cultivars. Breeding for resistance often is the most effective means of control. Sources of resistance or improved levels of tolerance for most diseases are available among the cultivated sunflower and the wild species of *Helianthus* [49].

Changes in the racial composition of certain pathogens have also been caused by the intro‐ duction of hybrids in commercial production, which are substantially more homogeneous with respect to the previous period when genetically heterogeneous open-pollinating varieties were grown.

Vear [50] recommended for efficient disease control in future breeding programs to combine vertical and horizontal resistance if available. If not, marker-assisted selection should be used to combine QTLs with different additive defense mechanisms [22].

Galina Pustovoit [51] evaluated new cultivars based on interspecific hybridization (*H. tuberosus* × cultivated sunflower) – Progress, October, Yubileyniy 60, and Novinka. Based on the results achieved in the field and by inoculation, the author concluded that the new cultivars possess group immunity, that is, resistance to downy mildew, rust, *Macrophomina*, *Phoma*, and broomrape.

To be successful in breeding for disease resistance, the sunflower breeder must be thoroughly acquainted with general principles of resistance breeding, major approaches to management of resistance genes, stability of sunflower resistance to certain pathogens, monitoring of interactions between the host (sunflower), pathogen and the environment, and resistance types (vertical and horizontal). Finally, he has to have an adequate germplasm at his disposal, select a method of breeding, and develop a strategy for achieving the desired goal [22].

The aim of this research is to review biotic stresses in sunflower, indicate their significance, and reveal the sources of resistance and methods of selection in order to achieve the desired goal.

#### *2.1.1. Downy mildew [Plasmopara halstedii (Farl.) Berl. et de Toni)]*

have achieved significant results in finding genes for resistance or high tolerance to certain diseases in wild species and incorporating them into cultivated sunflower genotypes possess‐

Wild sunflower species have been a valuable source of resistance genes for many of the common pathogens of the cultivated sunflower. The relative severity of individual diseases varies widely, depending on climate and host cultivars. Breeding for resistance often is the most effective means of control. Sources of resistance or improved levels of tolerance for most diseases are available among the cultivated sunflower and the wild species of *Helianthus* [49].

Changes in the racial composition of certain pathogens have also been caused by the intro‐ duction of hybrids in commercial production, which are substantially more homogeneous with respect to the previous period when genetically heterogeneous open-pollinating varieties were

Vear [50] recommended for efficient disease control in future breeding programs to combine vertical and horizontal resistance if available. If not, marker-assisted selection should be used

Galina Pustovoit [51] evaluated new cultivars based on interspecific hybridization (*H. tuberosus* × cultivated sunflower) – Progress, October, Yubileyniy 60, and Novinka. Based on the results achieved in the field and by inoculation, the author concluded that the new cultivars possess group immunity, that is, resistance to downy mildew, rust, *Macrophomina*, *Phoma*, and

to combine QTLs with different additive defense mechanisms [22].

ing high combining ability [22].

**Table 1.** The most common sunflower diseases

grown.

broomrape.

**Disease Pathogen**

600 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Downy mildew *Plasmopara halstedii* Broomrape *Orobanche cumana* White rot *Sclerotinia sclerotiorum* Stem canker *Diaporthe helianthi*

Rust *Puccinia helianthi* Phoma black stem *Phoma macdonaldii*

Verticillium wilt *Verticillium dahliae* Charcoal rot *Macrophomina phaseolina* White blister rust *Albugo tragopogonis* Fusarium wilt *Fusarium* spp. Rhizopus head rot *Rhizopus* spp.

Alternaria blight *Alternaria helianthi, A. helianthinficiens*

Virus *Sunflower chlorotic mottle virus*

Downy mildew [*Plasmopara halstedii* (Farl.) Berl. et de Toni)] occurs in all regions around the world in which sunflower is grown as a major oil crop. Downy mildew occurs with light intensity in years with a wet spring.

Downy mildew control was successfully maintained with dominant genes for a long period. This period roughly corresponds to the presence of only two races of downy mildew, the European race, controlled by the dominant gene *Pl1*, and North American, controlled by the *Pl2* gene. Unfortunately, changes took place in the past 14 years and there occurred a number of new races. These new races of downy mildew were registered in France, Hungary, USA, Argentina, and several other countries [22].

Viranyi [52] reported that the most detailed and up-to-date list of global distribution of *P. halstedii* pathogens has been compiled by Gulya [54] in a paper presented at the 2nd Interna‐ tional Downy Mildew Symposium, Kostelec, Czech Republic. In the accurate overview, he comprised as many as 34 pathotypes (races), an unbelievably high number considering the fact that in most sunflower-producing countries from just a few to 12 well-distinguished virulence phenotypes exist. Europe, France, Germany, and Spain reported the highest numbers but the pathogen is rather diverse in the USA, Canada, and in South Africa as well. Further‐ more, there are five *P. halstedii* pathotypes (300, 330, 710, 730, and 770) that are universally distributed globally, recorded from North and South America, Europe and Africa. Apart from the quantitative aspect of virulence, it is interesting to consider the dynamics of diversity as well, that is, the changes in a given region over time. In this respect, France leads with the highest number of new pathotypes arisen in the last 6–7 years [53].

Here, it should be mentioned that genes for resistance to the new races were quickly found in wild species and promptly transferred into genotypes of the cultivated sunflower [55]. An international set of differential lines has been made which makes it possible to determine which downy mildew races are present in a certain region. The set of differentials is supplemented with new lines as new downy mildew races occur [50].

The dynamism of changes in downy mildew races may be illustrated by the fact that, conclu‐ sive, with 2011, at least 18 downy mildew races have been determined in the world (100, 300, 304, 307, 314, 330, 700, 703, 704, 710, 711, 714, 717, 721, 730, 731, 770,...).

The testing of breeding materials by inoculation methods is in constant improvement and continuous progress. These issues have been dealt with by a large number of researchers, including Gulya *et al*. [56], Gulya *et al*. [57], Jouffret *et al.* [58], Tourvieille de Labrouhe *et al.* [59], Molinero-Demilly *et al.* [60], and others.

Tourvieille de Labrouhe *et al.* [61] reported that, in addition to major genes, nonrace-specific resistance contributes to the expression of resistance to downy mildew as well. The study also showed that the nonrace-specific resistance is inherited independently of major genes. Furthermore, Vear *et al.* [62] concluded that the inheritance of nonrace-specific resistance is under additive control. The authors reported that two QTLs may explain 42% variation in field reaction to downy mildew. This form of resistance was mapped as belonging to linkage groups 8 and 10. At the same time, they argued that this quantitative resistance is not related to any of the known major resistance gene clusters.

Also, Vear *et al.* [63] have developed a procedure for the development of new B-lines and parallel conversion into the *cms* form from source population. We should also mention here the procedure (scheme) of Vear *et al.* [64] for introgressing *Pl* genes into elite B-lines by backcrossing and simultaneous conversion into the *cms* form while performing resistance screening at the molecular level. This method significantly shortens the cycle of *Pl* gene introgression into elite lines.

According to Tourvieille de Labrouhe *et al.* [65] and Vear *et al.* [62], breeders should develop a strategy of simultaneous selection for nonspecific resistance and major gene resistance along with requisite use of molecular markers.

According to Seiler [49], complete resistance to the downy mildew pathogen was found in annual species *H. annuus, H. argophillus, H. debilis,* and *H. petiolaris* and perennial *H. decapetalus, H. divaricatus, H. eggertii, H. giganteus, H xlaetiflorus, H. mollis, H. nuttallii, H. scaberrimus, H. pauciflorus, H. salicifolius,* and *H. tuberosus* [66].

Diploid perennial species *H. divaricatus, H. giganteus, H. glaucophyllus, H. grosseserratus, H. mollis, H. nuttallii,* and *H. smithii* and their interspecific hybrids were resistant to downy mildew [67].

With the rapid improvement of molecular techniques and their use in plant pathology, new developments have opened new insights into research on fungal biology, detection technolo‐ gy, and genetics and host–pathogen interactions. For example, Hammer *et al.* [68] in Germany, using different approaches, were successful in detecting fungal structure from sunflower host tissues.

#### *2.1.2. White rot [Sclerotinia sclerotiorum (Lib.) de Bary]*

White rot is a major problem in countries with a humid climate or in years with an extremely wet summer. The fungus itself is polyphagous. It attacks over 360 plant species, which increases its variability and makes the selection for resistance difficult [69]. The major problem in the selection are the three types of the diseases (on the root, stem, and head) controlled by different mechanisms of resistance [11].

Sunflower stalk and head rot incident by *Sclerotinia sclerotiorum* (Lib.) de Bary is considered the most important disease of the crop in many parts of the world. Since cultural practices or fungicides are insufficient to control the disease, efforts are being made by breeders to develop resistant or tolerant cultivars. This may explain the dominance of publications dealing with various aspects of resistance [52].

The testing of breeding materials by inoculation methods is in constant improvement and continuous progress. These issues have been dealt with by a large number of researchers, including Gulya *et al*. [56], Gulya *et al*. [57], Jouffret *et al.* [58], Tourvieille de Labrouhe *et al.* [59],

Tourvieille de Labrouhe *et al.* [61] reported that, in addition to major genes, nonrace-specific resistance contributes to the expression of resistance to downy mildew as well. The study also showed that the nonrace-specific resistance is inherited independently of major genes. Furthermore, Vear *et al.* [62] concluded that the inheritance of nonrace-specific resistance is under additive control. The authors reported that two QTLs may explain 42% variation in field reaction to downy mildew. This form of resistance was mapped as belonging to linkage groups 8 and 10. At the same time, they argued that this quantitative resistance is not related to any

Also, Vear *et al.* [63] have developed a procedure for the development of new B-lines and parallel conversion into the *cms* form from source population. We should also mention here the procedure (scheme) of Vear *et al.* [64] for introgressing *Pl* genes into elite B-lines by backcrossing and simultaneous conversion into the *cms* form while performing resistance screening at the molecular level. This method significantly shortens the cycle of *Pl* gene

According to Tourvieille de Labrouhe *et al.* [65] and Vear *et al.* [62], breeders should develop a strategy of simultaneous selection for nonspecific resistance and major gene resistance along

According to Seiler [49], complete resistance to the downy mildew pathogen was found in annual species *H. annuus, H. argophillus, H. debilis,* and *H. petiolaris* and perennial *H. decapetalus, H. divaricatus, H. eggertii, H. giganteus, H xlaetiflorus, H. mollis, H. nuttallii, H. scaberrimus, H.*

Diploid perennial species *H. divaricatus, H. giganteus, H. glaucophyllus, H. grosseserratus, H. mollis, H. nuttallii,* and *H. smithii* and their interspecific hybrids were resistant to downy

With the rapid improvement of molecular techniques and their use in plant pathology, new developments have opened new insights into research on fungal biology, detection technolo‐ gy, and genetics and host–pathogen interactions. For example, Hammer *et al.* [68] in Germany, using different approaches, were successful in detecting fungal structure from sunflower host

White rot is a major problem in countries with a humid climate or in years with an extremely wet summer. The fungus itself is polyphagous. It attacks over 360 plant species, which increases its variability and makes the selection for resistance difficult [69]. The major problem in the selection are the three types of the diseases (on the root, stem, and head) controlled by

Molinero-Demilly *et al.* [60], and others.

602 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

of the known major resistance gene clusters.

with requisite use of molecular markers.

*pauciflorus, H. salicifolius,* and *H. tuberosus* [66].

*2.1.2. White rot [Sclerotinia sclerotiorum (Lib.) de Bary]*

different mechanisms of resistance [11].

introgression into elite lines.

mildew [67].

tissues.

When breeding sunflower for resistance to all three forms of *Sclerotinia* attack, it is necessary to combine two or three different tests [70].

Mancel and Shein [71] found that *Sclerotinia* isolates taken from different plant species differed in the degree of virulence. They also found that sunflower isolates that had been repeatedly subcultured in the laboratory were significantly less virulent than isolates recently obtained from sunflower.

When we discuss the three types of sunflower infection by *Sclerotinia*, it is easy to achieve high tolerance to the mid-stalk infection by selecting genotypes resistant to lice [11]. Young leaves of such genotypes are not injured by lice and therefore these plants avoid infections.

Using four different tests for the evaluation of *Sclerotinia* resistance (basal stem infection, ascospore, and oxalic acid injection into the back face of the head), Baldini *et al.* [72] found that the inbred line 28R was most tolerant to the basal stem and white head rot infections and it also showed the best performance in oxalate and culture filtrate tests, which indicated the presence of a specific resistance to oxalate.

Van Becelaere and Miller [73] tried different inoculation procedures for evaluation of resistance to *Sclerotinia* head rot. According to their results, the best method involved the spraying of heads at the beginning of flowering with 4 cm3 of a suspension of ascospores, which contained 4000 ascospores per milliliter, and covering the heads with brown paper bags immediately after inoculation. Measurements of inoculation could begin as early as 34 days after the inoculation.

Vear *et al.* [50] studied the virulence of 10 *Sclerotinia* isolates. They found differences in in vitro growth rate and sclerotia production as well as some highly significant isolate and genotype effects. They concluded that the available resistance in sunflower genotypes has partial, nonrace-specific, and horizontal characteristics and that it should be durable.

Using an in vitro screening test based on callus induction to evaluate *Sclerotinia* resistance, Drumeva *et al.* [74] found that the test allowed the identification of the breeding material with high to moderate resistance to the pathogen.

When developing inbred lines, sunflower breeders should take note of the results of Van Becelaere [75], who found that the general combining ability (GCA) effects of female lines were relatively larger than the GCA effects of male lines, which indicated that, at least in that particular research, the female lines had a greater influence on the resistance of the hybrids.

When considering the methods of selection for white rot tolerance, recurrent selection and pedigree method were found to produce the best results.

Vear *et al.* [63] used the pedigree method to select sunflower heads resistant to *Sclerotinia*. They applied the ascospore test on F2 and F4 plants and the mycelium test on F3 plants. Their results showed that in all cases there was a variation in the level of resistance among F3 families. The gains in relation to their parents ranged from 24 to 61%.

Vear *et al.* [76] applied 14 cycles of recurrent selection to a sunflower restorer population developed in 1978 and they obtained significant results. The mycelium test was used in the first three cycles and a combined test with a suspension of ascospores in the subsequent cycles. About 80% reduction of the infected area was achieved in the fourth cycle. In the 12th cycle, the latency index (a measure of incubation period) in the ascospore test was doubled. Simple regression provided the best relation with this cycle, indicating that further increase in the degree of tolerance was possible.

Christov [66] and Christov *et al.* [77] reported that higher-ploidy perennial species (hexaploid and tetraploid species) exhibited greater susceptibility than the diploids, with *H. glaucophyllus, H. divaricatus, H. salicifolius,* and *H. mollis* having the highest frequency of healthy plants. Tolerance to *Sclerotinia* was observed in the perennials *H. eggertii, H. pauciflorus,* and *H. smithii* and annuals *H. annuus, H. argophillus, H. petiolaris,* and *H. praecox* [78].

Interspecific hybrids based on *H. nuttallii, H. giganteus,* and *H. maximiliani* were reported to show resistance against stem infection by Henn *et al.* [79]. Miller and Gulya [80] developed four maintainer and four restorer oilseed lines with improved tolerance to *Sclerotinia* stalk rot. The inbred line HA 410 released by Miller and Gulya [80] derived from a wild perennial hexaploid, *H. pauciflorus (=rigidus*), had a moderate tolerance to stalk rot. *Sclerotinia* root rot tolerance was observed in perennials *H. mollis, H. nuttallii, H. resinosus,* and *H. tuberosus* [81].

Among the perennial species, resistance to *Sclerotinia* was observed in population of *H. tuberosus, H. divaricatus, H. hirsutus, H. maximiliani, H. mollis, H. nuttallii, H. occidentalis,* and *H. rigidus* (= *pauciflorus*) grown under natural infection conditions [82].

*Sclerotinia* head rot tolerance was observed in perennials *H. resinosus, H. tuberosus, H. decape‐ talus, H. grosseserratus, H. nuttallii,* and *H. pauciflorus* [83–85].

In the past decade, advances were made in the research of *Sclerotinia* resistance at the molecular level, particularly in the marker-assisted selection [86, 50, 62, and many others]. The new methods are expected to provide significant help to sunflower breeders [86].

#### *2.1.3. Sunflower rust (Puccinia helianthi Schw.)*

Rust is the second most important sunflower disease considering its global distribution. The disease causes economic loargophyllussses in sunflower production in North and South America, Australia and Africa. Based on our own observations, rust is present in several countries in Asia (China, India, Iran, Kazakhstan, and others), but its racial composition has not been determined yet. Fortunately for Europe, the local rust population is fairly stable. Rust races were studied most extensively in North America. Sackston [87] determined four North American races, 1, 2, 3, and 4. Race 4 was identified by Yang [88] and race 6 by Lambrides and Miller [89].

Antonelli [90] and Senetinner *et al.* [91] studied sunflower resistance to an Argentinean rust isolate, clone 340, and found that the lines MP 447, MP 444, and LC 74/74-20620 were resistant to it and that the resistance was controlled by a single dominant gene.

Hugues *et al.* [92] studied the occurrence and distribution of rust in Argentina in the period 1982–2008. Their results indicated that resistant cultivars were stable in terms of rust resistance. They also concluded that a single rust pathotype existed in central and southern sunflowergrowing regions of Argentina, which was in contrast to previous studies.

In Africa, the determination of rust races in sunflower was done only in Mozambique. Using differential lines from Canada and USA, Vicente and Zazzerini [93] found that the rust race 4 was present in Mozambique.

In Europe, rust has been studied on a limited scale. Most of the work had been done at VNIIMK, Krasnodar. Studying various methods of inoculation by rust, Galina Pustovoit and Slyusar [94] concluded that growing a mixture of resistant genotypes in spatial isolation completed by selection of resistant plants was the most appropriate method.

Miller *et al.* [95] tested 343 genotypes for resistance to rust and found that 12 genotypes were resistant to race 4. The authors also found that the lines HA-R1, HA-R3, HA-R4, HA-R34, and 647-1 shared the same locus, *R4*, while the line HA-R2 had a different one that was named R4.

Kochman and Goulter [96] proposed a system for identification of rust races in sunflower, and examined the slow-rusting phenomenon and resistance gene pyramiding to control sunflower rust.

Sendall *et al.* [97] studied the diversity of *Puccinia helianthi* pathosystem in sunflower in Australia at the molecular level and found a set of 24 lines and determined putative resistance genes.

Regarding the methods of artificial inoculation, Gulya and Maširević [98] provided a detailed description of inoculation techniques for evaluating sunflower resistance to rust under laboratory conditions (greenhouse experiments) as well as under field conditions. They also ranked the differential lines in three sets: set one (S-37-388, CM90RR, and MC29), set two (P-386, HA-R1, and HA-R2), and set three (R3-HA, HA-R4, and R4-HA).

Wild species of the genus *Helianthus* are a rich gene pool for further identification of resistance genes and their use to forestall the emergence of new races of *Puccinia helianthi*.

#### *2.1.4. Stem canker (Phomopsis) Diaporthe helianthi*

Vear *et al.* [63] used the pedigree method to select sunflower heads resistant to *Sclerotinia*. They applied the ascospore test on F2 and F4 plants and the mycelium test on F3 plants. Their results showed that in all cases there was a variation in the level of resistance among F3 families. The

Vear *et al.* [76] applied 14 cycles of recurrent selection to a sunflower restorer population developed in 1978 and they obtained significant results. The mycelium test was used in the first three cycles and a combined test with a suspension of ascospores in the subsequent cycles. About 80% reduction of the infected area was achieved in the fourth cycle. In the 12th cycle, the latency index (a measure of incubation period) in the ascospore test was doubled. Simple regression provided the best relation with this cycle, indicating that further increase in the

Christov [66] and Christov *et al.* [77] reported that higher-ploidy perennial species (hexaploid and tetraploid species) exhibited greater susceptibility than the diploids, with *H. glaucophyllus, H. divaricatus, H. salicifolius,* and *H. mollis* having the highest frequency of healthy plants. Tolerance to *Sclerotinia* was observed in the perennials *H. eggertii, H. pauciflorus,* and *H.*

Interspecific hybrids based on *H. nuttallii, H. giganteus,* and *H. maximiliani* were reported to show resistance against stem infection by Henn *et al.* [79]. Miller and Gulya [80] developed four maintainer and four restorer oilseed lines with improved tolerance to *Sclerotinia* stalk rot. The inbred line HA 410 released by Miller and Gulya [80] derived from a wild perennial hexaploid, *H. pauciflorus (=rigidus*), had a moderate tolerance to stalk rot. *Sclerotinia* root rot tolerance was observed in perennials *H. mollis, H. nuttallii, H. resinosus,* and *H. tuberosus* [81].

Among the perennial species, resistance to *Sclerotinia* was observed in population of *H. tuberosus, H. divaricatus, H. hirsutus, H. maximiliani, H. mollis, H. nuttallii, H. occidentalis,* and *H.*

*Sclerotinia* head rot tolerance was observed in perennials *H. resinosus, H. tuberosus, H. decape‐*

In the past decade, advances were made in the research of *Sclerotinia* resistance at the molecular level, particularly in the marker-assisted selection [86, 50, 62, and many others]. The new

Rust is the second most important sunflower disease considering its global distribution. The disease causes economic loargophyllussses in sunflower production in North and South America, Australia and Africa. Based on our own observations, rust is present in several countries in Asia (China, India, Iran, Kazakhstan, and others), but its racial composition has not been determined yet. Fortunately for Europe, the local rust population is fairly stable. Rust races were studied most extensively in North America. Sackston [87] determined four North American races, 1, 2, 3, and 4. Race 4 was identified by Yang [88] and race 6 by Lambrides and

*smithii* and annuals *H. annuus, H. argophillus, H. petiolaris,* and *H. praecox* [78].

*rigidus* (= *pauciflorus*) grown under natural infection conditions [82].

methods are expected to provide significant help to sunflower breeders [86].

*talus, H. grosseserratus, H. nuttallii,* and *H. pauciflorus* [83–85].

*2.1.3. Sunflower rust (Puccinia helianthi Schw.)*

Miller [89].

gains in relation to their parents ranged from 24 to 61%.

604 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

degree of tolerance was possible.

In the past three decades, *Phomopsis* has become the most destructive disease on the global scale. Its large-scale occurrence was first registered in the Vojvodina Province (Serbia) and Romania in 1980, when it caused large economic damage to sunflower production. Soon afterwards, it was registered in most sunflower-growing countries in Europe (France, Hungary, Slovakia, Bulgaria, Ukraine, Russia, and Italy). In the early 1980s, its presence was reported in the USA, Canada, Argentina, Uruguay, Australia, Iran, and some other coun‐ tries [22].

The first significant results in sunflower breeding for resistance to *Phomopsis* were achieved in Serbia and Romania.

Škorić [99] reported that of 4000 inbred lines and 2000 experimental hybrids, only four lines exhibited field resistance to *Phomopsis*. Two of these lines had been derived from interspecific hybrids (cultivated sunflower × *H. tuberosus*): one was obtained from a cross of *H. argophyl‐ lus* × Armavirski 9344 and the restorer line SNRF-69 was derived from a local population from Hungary.

Based on extensive research, Vrânceanu *et al.* [100] found that the sunflower resistance to *Phomopsis* is of the horizontal type and that it is positively correlated with the stay-green phenomenon. The authors reported that, of all Romanian hybrids, Select has the highest degree of tolerance to *Phomopsis*.

Škorić [99] found that three female lines (Ha-22, Ha-74, and Ha-BCPL) and the restorer line SNRF-6 are field resistant to *Phomopsis*. Resistance was transferred to the hybrids NS-H-43, NS-H-44, and H-NS-44 developed from these lines. The same author also reported that *Phomopsis* resistance is positively correlated with *Macrophomina* and *Phoma* resistance as well as with drought tolerance.

Vrânceanu *et al.* [101] concluded that partial dominance is expressed in the inheritance of *Phomopsis* resistance in some cases, while additive inheritance is much more frequent. The same authors found that the stay-green stem at the ripening stage is positively correlated with *Phomopsis* resistance.

Much work has been done lately on the use of molecular markers in breeding for *Phomopsis* resistance.

Studying recombinant inbred lines derived by crossing LR4-17 (resistant) with HA89 (suscep‐ tible) at the molecular level, Langar *et al.* [102, 103] concluded that unlinked segments carried major QTLs for different components of resistance, and that the resistances of leaves and stems could be pyramided with a marker-assisted selection.

Molecular studies on the intraspecific diversity of this fungus using intergenic spacer sequence analysis revealed a high homology among French/Yugoslavian and among Italian isolates [104]. The phylogenetic tree obtained from the aligned data revealed three separate groups. The analysis also showed that all isolates originating from countries with regular and severe outbreaks of the disease (e.g., France, Yugoslavia, etc.) formed a well-defined taxon with relatively low variability compared with isolates from Italy where the disease seldom occurs. In another paper, Rekab *et al.* [105] pointed out a polyphyletic nature of this fungus.

Škorić [99] and Dozet [106] reported high levels of resistance to *Phomopsis* in *H. maximiliani*, *H. hirsutus*, *H. pauciflorus*, *H. mollis*, *H. resinosus,* and *H. tuberosus*.

Interspecific hybrids based on *H. eggertii* and *H. smithii* showed high tolerance to *Phomopsis* in Bulgaria [107].

Christov [78] identified annuals *H. annuus*, *H. argophyllus*, and *H. debilis* and perennials *H. pauciflorus*, *H. glaucophyllus,* and *H. eggertii* as potential sources of *Phomopsis* brown stem canker resistance, based on field screening in Bulgaria.

Nikolova *et al.* [108] reported resistance to stem canker in progenies of interspecific hybrids of perennial *H. pumilus*. Resistance to *Phomopsis* was reported in interspecific hybrids derived from *H. argophyllus*, *H. deserticola*, *H. tuberosus*, and *H. xlaetiflorus* [109].

Complete resistance to *Phomopsis* was reported in interspecific hybrids of *H. salicifolius* by Encheva *et al.* [110] and Škorić [22].

State research and private companies have developed a rich germplasm for *Phomopsis* resistance.
