Advances on Pod and Stem Blight of Soybean (Diaporthe phaseolorum var. sojae and Phomopsis longicolla) in Argentina

Natural dispersal

Soybean seeds and crop residues were formerly recognized as sources of inoculum for D. phaseolorum var. sojae and Phomopsis spp. [11,20]. All colonized tissues are potential sources of primary inoculum, although the bulk of primary inoculum appears to originate from over-wintered or over-summered crop debris [21,22].

Seedborne spread

An effective way to introduce Phomopsis into new areas fungusfree is the movement of infected potentially seeds [1,21]. However, seed-borne inoculum, even on 77% of incidence; was not related to infections on either seedlings or mature plants in fields with continuous soybean or corn-soybean rotation, which indicated that seeds were a minor inoculum source [21]. These results allowed to infer that soybean infected residues with Phomopsis could be the major inoculum sources for pod and stem blight [20,21,23]. Similar results were obtained in Santa Fe, Argentina where continuoussoybean rotation increased the incidence of Phomopsis when compared with corn-soybean rotation [24].

Economic impact

Human consumption of cooked soybeans and soy flour may help alleviate global protein deficiency, especially in high populating developing countries [25]. Therefore, soybean seed quality, judged by germination and general appearance of the seed has been relevant aspects to consider in many of the soybean-growing areas of the world. However, tropical areas with high rainfall and temperature favor the development of seed-borne Diaporthe phaseolorum var. sojae (Phomopsis sojae) and P. longicolla, their broad distribution and high frequency deserve careful attention. Formerly, pod and stem blight was cited as a common disease in Illinois, Iowa, Indiana, USA, and Ontario, Canada [26-29]. Later, in Midwestern Maryland and Delaware this fungus was considered the predominant organism associated with low seed quality [6]. Louisiana, bio-assayed for internally borne pathogens, showed 97% infection with D. phaseolorum var. sojae [5]. In Illinois, seeds of soybean with symptoms of stem pod blight infection were smaller in size and volume, lower in density, produced lower quality oil and flour and had lower viability and durability than symptomless seeds. Oil from infected seeds had rancid off smell, and a high peroxide value, indicating oil deterioration [30]. Phomopsis seed decay and stem canker constitute diseases that more severely affect soybean seed quality and yield, and they are present in almost every region of soybean production in the world [22]. In South America P. sojae was identified on naturally infected soybean plants in Argentina (Memoria anual 1979), in Brazil (Castro and Kimati, 1981; Almeida, 1981; Berger and Hinson, 1984), and pathogenicity was confirmed in Venezuela (Sanabria de Albarracin, 1993). Other studies also reported the importance of Phomopsis spp. (P. sojae, P. longicolla, Phomopsis spp.) as seed-borne pathogen associated to green seed soybean (eda mamé) [31], fruits and seeds of native forest species [32] and the epidemiological role the infection and colonization progress in plant tissues by this fungus in the core of soybean area [3,33].

Effect on seed quality

Infection of soybean seeds by the Diaporthe/Phomopsis complex decreases physiological quality and viability leading to significant losses in germination [34]. Oil extracted from infected seeds typically has a lower quality, characterized by a rancid smell and a high peroxide value, which indicates oil deterioration [30,35]. And the quality of the produced flour was also diminished. For horticultural use such as vegetable soybean or edamame, infected pods and seeds are considered unsuited for consumption due to these qualitative defects [17,31,36], consequently, the presence of these pathogens impacts not only on the seed’s health but also on its overall weight, industrial utility, and commercial value [17,22].

Phytosanitary risk

In Argentina, Phomopsis longicolla (Plo) and P. phaseoli var. sojae (Pps) (Teleomorph Diaporthe phaseolorum var. sojae, Dps) are the primary species associated with soybean, as well as horticultural varieties intended for fresh consumption or edamame [31]. Their interaction leads to Stem and Pod Blight and Seed Decline (TTVyS), drastically reducing seed weight and quality. While Pps possesses a known teleomorph (sexual) stage, Plo has only been observed in its anamorphic form. Both species utilize stubble and seeds as primary inoculum sources, and their biological plasticity allows them to colonize diverse agro-ecosystems, even affecting the seed germination of tree species like Schinopsis balansae [32,33]. The biological plasticity of this complex has facilitated its expansion into diverse agro-ecosystems, the transmission through seeds and long-distance transport increasing the epidemiological risk; thus constituted the main pathways to introduce inoculum into plots previously free of the disease [17]. Molecular studies revealed significant genetic variability within the complex, favoring the emergence of new physiological races with enhanced parasitic capacity. This diversity in both fungal and plant germplasm complicates disease management. Consequently, Argentine research has pivoted toward characterizing specific sources of resistance to Stem Blight.

Identification based on morphological and molecular tools

To resolve taxonomic ambiguities, an approach combining morphological characterization with molecular tools was employed to evaluate Argentine isolates of Plo and Pps from different environments. For morphological analysis: Macroattributes (colony texture, stroma/pycnidia distribution) and micro-attributes (conidia, asci, and ascospore dimensions) were evaluated. Whilst several molecular analyses validated the identity of isolates where morphology was limited. Thus, 12 isolates were selected and categorized into four taxa: Ten as P. longicolla; one as D. phaseolorum var. sojae (or Pps); and to use as experimental control: one D. phaseolorum var. caulivora (Dpc) and two D. phaseolorum var. meridionalis (Dpm). The combination of phenotypic and molecular tools is essential for the accurate identification of species within the DP complex. These findings enhance the understanding of fungal plasticity, host expansion mechanisms, and biological relationships (such as (homo or heterothallism and hybridization), providing a critical foundation for managing genetic variability and preserving fungal biodiversity in agricultural systems [33].

Pathogenic diversity of Phomopsis sp. causal agent of soybean (Glycine max) stem and pod blight

Molecular studies applied on Dps and Plo demonstrated a remarkable genetic variability within this fungal group [22], that gave rise to the appearance of new physiological races [8,47]. Resistant genotypes to SPB-SD were obtained by breeding programs worldwide, but not particularly to SB. Therefore, although in Argentina it was possible to characterize and select some potential sources of resistance [48], it was relevant deepening the study of these diseases. In this case, the objective was to study genetic and pathogenic variability of Phomopsis in interactions with cultivars of varied resistance or susceptibility, and to study the association between molecular and pathogenic profiles in the analyzed interactions. The hypothesis was that the existence of genetic variability in both fungus and plant materials increases the biodiversity in specific reactions during the development of SB. For contrasting this hypothesis, a new approach of the widely known statistical methods GGE Biplot was proposed for measuring the correlation among pathogenic attributes (phenotypic expression of the plant pathology) and molecular markers. Six isolates of Plo and one of Pps were inoculated to six soybean cultivars. Genetic variability in the fungi was evaluated at the molecular level by RAPD and ITS markers and at the phenotypic level by their pathogenic performance through the severity (S%) of the pathology caused in soybean cultivars. Molecular characterization separated the Plo isolates from the Pps isolate. Specific interaction between each isolate-cultivar combination evidenced differential pathogenic performances in respect to resistance/susceptibility. Biplot GGE analysis allowed visualization of specific interactions where certain isolates express their maximum virulence on specific cultivars, demonstrating that genetic variability in both fungal and plant germplasm is associated with the diversity of specific reactions during disease development. Using this tool, specific polymorphic fragments (generated by the OP-AA01 primer) were identified, the presence or absence of which correlated with the ranking of Phomopsis isolates that cause SB in G. max, differentiated by severity, detecting molecular markers potentially associated with virulence [33].

Inheritance of resistance genes to SSB caused by Plo

In recent years, some soybean genotypes carrying Rpsd genes that confer resistance to soybean seed decay, caused by the same agents that cause SSB, have been reported. However, it was not known whether the Rpsd genes were also effective for Soybean Stem Disease (SSB). Therefore, in this case, the objective was the characterization of various soybean genotypes, carriers of Rpsd genes and others of interest, to evaluate their behavior against local strains of Phomopsis (Pps and Plo) that caused SSB, detecting eventual resistance genes (Rpsb) and determining their mode of inheritance. From these parents, crosses between RxS and RxR genotypes (Ge) were carried out during two planting seasons, 2015/16 and 2016/17. Of a total of 203 hybridizations performed (RxS and RxR), 43 fertile combinations producing pods and seeds were obtained, corresponding to a 21% cross effectiveness [49]. To identify truly hybrid individuals (with heterozygous complement) and ensure the accuracy of the resulting segregating generations, SNP-type molecular markers were used to evaluate the F1. Due to their robustness and greater accessibility thanks to reduced costs, SNP markers are currently the most widely used and promising class of markers. Based on the importance and progress achieved by applying these biotechnological tools to plant breeding, this thesis proposed the early and innovative application of specific molecular markers to characterize the first generation (F1), making the selection process more efficient from the beginning of soybean breeding work [33]. The use of SNP molecular markers in the initial stages of selection and genetic improvement allowed for obtaining F1 individuals who’s heterozygous and hybrid makeup was molecularly validated, ensuring safe progress in subsequent segregating generations [50].

Analyzing the SNP molecular characterization of each parental pair yielded information on four interesting aspects: the degree of polymorphism between parents, residual heterozygosity, the conversion of an allele to one of the parents (maternal effect), and the advantages and limitations of characterizing the first generation (F1) solely through observation of morphological traits [51]. Regarding the degree of polymorphism detected between parents, it emerged that the germplasm of some genotypes was more closely related to each other, as observed, for example, in Ge(1) and Ge(2), which are considered almost isolines, and some authors still debate whether they are the same genotype, where out of 1100 amplified SNPs, only 1 SNP was polymorphic for both. Meanwhile, other more divergent genotypes allowed the identification of up to 405 polymorphic SNPs between the parents, as in the case of Ge (1) and Ge (5). Regarding the residual heterozygosity detected in the parents through the loci characterized by polymorphic SNPs for 10 parents, it was observed that the Ge (6) and Ge (4) genotypes, which are stabilized cultivars used in various breeding programs, showed practically no loci in a state of residual heterozygosity (2 and 1, respectively). While the Ge (3) genotype registered 22 out of 564 residual heterozygous events, this may be due to a problem with seed purity, which could be associated with seed handling; however, in none of the cases it was higher than 4%. Likewise, when characterizing the F1 individuals, in a few cases a proportion of non-heterozygous loci were observed, which duplicated one of the alleles of one of the parents. Although this study observed a low proportion of loci like maternal parent, a phenomenon known as the “maternal effect” or gene conversion heterozygous genotypes, individuals with this percentage exceeding 10-12% were excluded [52-56]. A particular case was found for the crosses resulting from the white-flowered Ge(6) and purple-flowered Ge(1) genotypes, where the three F1 individuals from such a cross (Ge(6) x Ge(1).1A; Ge(6) x Ge(1).1B; Ge(6) x Ge(2).2), molecularly validated as heterozygous hybrids, presented white flowers as the maternal genotype Ge(6), contrary to what was expected based on the molecular analysis using SNPs [57]. This could be due to a residual heterozygosity effect from the paternal genotype Ge (1), in which, of the 1224 total SNPs, 920 amplified for this genotype, 848 were found in homozygosity and 72 in heterozygosity [10]. Within these 72 heterozygous SNPs, 6 were located on chromosome 13, the site of the allele coding for flower color (dominant purple W1, recessive white w1) [58].

The results obtained demonstrated that the additional and complementary application of molecular markers, combined with classical controls (morphological and structural-histological), provides rigor and relevant additional information about each of the parents involved in the crosses, enriching and strengthening the results and their biological significance. However, it is important to note that this same protocol simultaneously generated a significant reduction in diversity compared to the numerous original combinations and their respective RxS, RxR, and reciprocal crosses [59]. Thus, one of the proposed objectives has been met: to have molecularly validated F₁ populations and their respective heterozygous and hybrid parents from the first filial generation (F₁), that is, from the F₁ individuals, and to allow for safe progress in the segregating F₂ populations and their respective F₃ generations and F_2:3 families. Through inoculations, the reaction against SSB of the parents, the F₁, F₂ individuals and the F₃ plants distributed in the F_2:3 families (Progeny Tests) were characterized [60]. Through the observed phenotypic ratios, it was possible to infer the expected genotypic ratios in the F₂ parents, allowing the identification of the first SSB resistance gene (Rpsb1) to SSB, carried by one of the resistant genotypes, without ruling out the possibility of carrying other associated genes [61]. By evaluating Resistance / Susceptibility as a dichotomy qualitative trait, different types of epistatic interactions were detected in each different cross.

The preliminary results obtained by classic genetic improvement and molecular assistance contribute and strengthen the current studies for identifying resistance genes (Rpsb) to SSBPlo and their inheritance way. It was also demonstrated that stalk resistance (PSB) is genetically independent of the Rpsd genes linked to Seed Decay (SD). These advances provided an understanding about the effectiveness of the strategies applied and perspectives of plant improvement aimed at incorporating resistance to diseases in soybean crops.

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