Discriminant haplotypes of avirulence genes of Phytophthora sojae lead to a molecular assay to predict phenotypes

The soybean–Phytophthora sojae interaction operates on a gene-for-gene relationship, where the product of a resistance gene (Rps) in the host recognizes that of an avirulence gene (Avr) in the pathogen to generate an incompatible reaction. To exploit this form of resistance, one must match with precision the appropriate Rps gene with the corresponding Avr gene. Currently, this association is evaluated by phenotyping assays that are labour-intensive and often imprecise. To circumvent this limitation, we sought to develop a molecular assay that would reveal the avirulence allele of the seven main Avr genes (Avr1a, Avr1b, Avr1c, Avr1d, Avr1k, Avr3a, and Avr6) in order to diagnose with precision the pathotypes of P. sojae isolates. For this purpose, we analysed the genomic regions of these Avr genes in 31 recently sequenced isolates with different virulence profiles and identified discriminant mutations between avirulence and virulence alleles. Specific primers were designed to generate amplicons of a distinct size, and polymerase chain reaction conditions were optimized in a final assay of two parallel runs. When tested on the 31 isolates of known virulence, the assay accurately revealed all avirulence alleles. The test was further assessed and compared to a phenotyping assay on 25 isolates of unknown virulence. The two assays matched in 97% (170/175) of the interactions studied. Interestingly, the sole cases of discrepancy were obtained with Avr3a, which suggests a possible imperfect interaction with Rps3a. This molecular assay offers a powerful and reliable tool to exploit and study with greater precision soybean resistance against P. sojae.     

Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, as populations of P. sojae are highly diverse and quick to adapt, and can be overcome 8–15 years after deployment. Thus, it is important to identify novel Rps genes for development of resistant soybean cultivars. PI 567139B is a soybean landrace carrying excellent resistance to nearly all predominant P. sojae races in Indiana. A mapping population consisting of 245 F₂ individuals and 403 F_2:3 families was developed from a cross between PI 567139B and the susceptible cultivar ‘Williams’, and used to dissect the resistance carried by PI 567139B. We found that the resistance in PI 567139B was conferred by two independent Rps genes, designated RpsUN1 and RpsUN2. The former was mapped to a 6.5 cM region between SSR markers Satt159 and BARCSOYSSR_03_0250 that spans the Rps1 locus on chromosome 3, while the latter was mapped to a 3.0 cM region between BARCSOYSSR_16_1275 and Sat_144, approximately 3.0–3.4 cM upstream of Rps2 on chromosome 16. According to the ‘Williams 82’ reference genome sequence, both regions are highly enriched with NBS-LRR genes. Marker assisted resistance spectrum analyses of these genes with 16 isolates of P. sojae, in combination with the mapping results, suggested that RpsUN1 was likely to be a novel allele at the Rps1 locus, while RpsUN2 was more likely to be a novel Rps gene.     

Novel quantitative trait loci for partial resistance to Phytophthora sojae in soybean PI 398841

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is one of the most severe soybean [Glycine max (L.) Merr] diseases in the USA. Partial resistance is as effective in managing this disease as single-gene (Rps gene)-mediated resistance and is more durable. The objective of this study was to identify quantitative trait loci (QTL) associated with partial resistance to P. sojae in PI 398841, which originated from South Korea. A population of 305 F_7:8 recombinant inbred lines derived from a cross of OX20-8 × PI 398841 was used to evaluate partial resistance against P. sojae isolate C2S1 using a tray test. Composite interval mapping using a genome-wide logarithm of odd (LOD) threshold detected three QTL on chromosomes 1, 13, and 18, which individually explained 4–16 % of the phenotypic variance. Seven additional QTL, accounting for 2–3 % of phenotypic variance each, were identified using chromosome-wide LOD thresholds. Seven of the ten QTL for resistance to P. sojae were contributed by PI 398841. Seven QTL co-localized with known Rps genes and previously reported QTL for soil-borne root pathogens, isoflavone, and seed oil. Three QTL on chromosomes 3, 13, and 18 co-localized with known Rps genes, but PI 398841 did not exhibit an Rps gene-mediated resistance response following inoculation with 48 different isolates of P. sojae. PI 398841 is potentially a source of novel genes for improving soybean cultivars for partial resistance to P. sojae.     

Peroxidase Activity in Soybeans Following Inoculation with Phytophthora sojae

The effects of race-specific resistance as conditioned by Rps genes (rps, Rps1-k, Rps2, Rps3, Rps6) in two genetic backgrounds (Williams & Harosoy) on accumulation of soluble peroxidases were determined by a soybean peroxidase capture assay (SPCA) after inoculation with P. sojae races 2, 7, or 25. Peroxidase activity increased in all isolines during the 72 h after inoculation, but reactions varied depending on time after inoculation, genetic background, Rps gene and P. sojae race. Peroxidase activity was higher in race-specific resistant than in susceptible reactions at 72 h. after inoculation, except for plants with the Rps2 gene which confers a unique form of root resistance in addition to the whole plant race-specific resistance. Williams isolines had larger increases in peroxidase activity than Harosoy isolines when data were averaged across Rps genes, and was most evident when plants were inoculated with race 2. When soybeans were inoculated with race 7 Rps1-k resistant plants had the highest increase in peroxidase activity, but Rps2 susceptible plants had a significantly higher peroxidase activity than plants with rps, Rps3, and Rps6 that were also susceptible. Results from inoculations with race 25 were somewhat different, Rps2 resistant plants had the highest increase in peroxidase activity; however, plants with the Rps3 or Rps6 gene that were also resistant did not have a significantly higher peroxidase activity than susceptible plants with the rps or Rps1-k gene.     

Selection and Characterization of Inhibitor-Resistant Mutants of Phytophthora sojae

Bhat, R. G., and Schmitthenner, A. F. 1993. Selection and characterization of inhibitor-resistant mutants of Phytophthora sojae. Experimental Mycology 17, 109-121. Selectable markers, resistance to metalaxyl (MEX) and p -fluorophenylalanine (FPA), were induced in Phytophthora sojae races by treating zoospores with N -methyl-N′-nitro-N′-nitrosoguanidine. Calcium treatment enhanced the percentage encystment of zoospores. MEX-resistant (MEX^r) and FPA-resistant (FPA^r) mutants were selected on a lima bean agar medium containing MEX (10 μg/ml) and FPA (50 μg/ml), respectively. A number of single inhibitor-resistant mutants were obtained. There was variation in in vitro growth of mutants. Virulence of mutants was evaluated by hypocotyl inoculation of soybean cultivars with Rps 1 (Amsoy 71), Rps1-c (Williams 79), Rps1-k (Williams 82), or rps1 (Williams) alleles. It was observed that the 23% of the MEX^r and 33% of the FPA^r mutants had a different virulence pattern than the original cultures.     

Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, <Emphasis Type="Italic">RpsHC18</Emphasis>, in soybean

Key message

A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS–LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed.

Abstract

Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F_2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites–leucine-rich repeat (NBS–LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.

     

Pathotype and SSR variation in Phytophthora sojae from the Argentinean Pampas

Genetic variation in Phytophthora sojae was compared between northern (NPS) and southern (SPS) Pampeana sub-regions, in the core soya bean region of Argentina. Ninety-three isolates from plants and soil were evaluated in the present study. The pathotype for each isolate was determined using the hypocotyl technique on a set of eight differentials. For neutral genetic diversity evaluation, eight SSRs markers were utilized. Thirty pathotypes were determined, including 19 in SPS and 23 in NPS. The number of isolates for each locality and region was unevenly distributed. Pathotype variation was high for P. sojae samples compared among fields and within isolates from single fields. SSR variation studies resulted in an elevated haplotype composition, partitioned at all, but the regional levels. P. sojae populations from Argentina have evolved towards a complex epidemiological scenario, with no evidence of geographic correlation at the pathotypic nor the neutral genetic level. It is likely that future works aiming at elucidating the evolution of pathogenesis in this species will need to include non-neutral markers, specifically associated to avr loci. The results of the present work are considered a useful contribution to the adequate and sustainable management of this pathogen in the main soya bean area of Argentina.     

Genetic mapping and development of co-segregating markers of <Emphasis Type="Italic">RpsQ</Emphasis>, which provides resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean

Key message

The RpsQ Phytophthora resistance locus was finely mapped to a 118-kb region on soybean chromosome 3. A best candidate gene was predicted and three co-segregating gene markers were developed.

Abstract

Phytophthora root rot (PRR), caused by Phytophthora sojae, is a major threat to sustainable soybean production. The use of genetically resistant cultivars is considered the most effective way to control this disease. The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance, with a distinct resistance phenotype, following inoculation with 36 Chinese P. sojae isolates. Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene. This gene locus was designated as RpsQ and mapped to a 118-kb region between BARCSOYSSR_03_0165 and InDel281 on soybean chromosome 3, and co-segregated with Insert11, Insert144 and SNP276. Within this region, there was only one gene Glyma.03g27200 encoding a protein with a typical serine/threonine protein kinase structure, and the expression pattern analysis showed that this gene induced by P. sojae infection, which was suggested as a best candidate gene of RpsQ. Candidate gene specific marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3. Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1 (RpsQ) and Ludou 4 (Rps9). All other cultivars carrying Rps genes on chromosome 3 produced different PCR products, which all lacked a 144-bp fragment present in Qichadou 1 and Ludou 4. The phenotypes of the analyzed cultivars combined with the physical position of the PRR resistance locus, candidate gene analyses, and the candidate gene marker test revealed RpsQ and Rps9 are likely the same gene, and confer resistance to P. sojae.

     

Genetic analysis and fine mapping of RpsJS,a novel resistance gene to Phytophthora sojae in soybean [Glycine max (L.) Merr.]

Key message

We finely map a novel resistance gene ( RpsJS ) to Phytophthora sojae in soybean. RpsJS was mapped in 138.9 kb region, including three NBS-LRR type predicted genes, on chromosome 18.

Abstract

Phytophthora root rot (PRR) caused by Phytophthora sojae (P. sojae) has been reported in most soybean-growing regions throughout the world. Development of PRR resistance varieties is the most economical and environmentally safe method for controlling this disease. Chinese soybean line Nannong 10-1 is resistant to many P. sojae isolates, and shows different reaction types to P. sojae isolates as compared with those with known Rps (Resistance to P. sojae) genes, which suggests that the line may carry novel Rps genes or alleles. A mapping population of 231 F₂ individuals from the cross of Nannong 10-1 (Resistant, R) and 06-070583 (Susceptible, S) was used to map the Rps gene. The segregation fits a ratio of 3R:1S within F₂ plants, indicating that resistance in Nannong 10-1 is controlled by a single dominant gene (designated as RpsJS). The results showed that RpsJS was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by SSR (simple repeat sequences) markers BARCSOYSSR_18_1859 and SSRG60752K at a distance of 0.9 and 0.4 cm, respectively. Among the 14 genes annotated in this 138.9 kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950 and Glyma18g51960) are the nucleotide-binding site and a leucine-rich repeat (NBS-LRR) type gene, which may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on marker-assisted resistance spectrum analyses of RpsJS and the mapping results, we inferred that RpsJS was a novel gene or a new allele at the Rps4, Rps5 or Rps6 loci.     

Genetic characterization and fine mapping of the novel Phytophthora resistance gene in a Chinese soybean cultivar

Phytophthora root rot (PRR), caused by Phytophthora sojae Kaufmann & Gerdemann, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.]. Deployment of resistance genes is the most economical and effective way of controlling the disease. The soybean cultivar ‘Yudou 29’ is resistant to many P. sojae isolates in China. The genetic basis of the resistance in ‘Yudou 29’ was elucidated through an inheritance study and molecular mapping. In response to 25 P. sojae isolates, ‘Yudou 29’ displayed a new resistance reaction pattern distinct from those of differentials carrying known Rps genes. A population of 214 F_2:3 families from a cross between ‘Jikedou 2’ (PRR susceptible) and ‘Yudou 29’ was used for Rps gene mapping. The segregation fit a ratio of 1:2:1 for resistance:segregation:susceptibility within this population, indicating that resistance in ‘Yudou 29’ is controlled by a single dominant gene. This gene was temporarily named RpsYD29 and mapped on soybean chromosome 03 (molecular linkage group N; MLG N) flanked by SSR markers SattWM82-50 and Satt1k4b at a genetic distance of 0.5 and 0.2 cM, respectively. Two nucleotide binding site-leucine rich repeat (NBS-LRR) type genes were detected in the 204.8 kb region between SattWM82-50 and Satt1k4b. These two genes showed high similarity to Rps1k in amino acid sequence and could be candidate genes for PRR resistance. Based on the phenotype reactions and the physical position on soybean chromosome 03, RpsYD29 might be a novel allele at, or a novel gene tightly linked to, the Rps1 locus.     

Biological Control of Phytophthora Root Rots on Alfalfa and Soybean with Streptomyces

A collection of 53 antibiotic-producing Streptomyces isolated from soils from Minnesota, Nebraska, and Washington were evaluated for their ability to inhibit plant pathogenic Phytophthora medicaginis and Phytophthora sojae in vitro. Eight isolates having the greatest pathogen-inhibitory capabilities were subsequently tested for their ability to control Phytophthora root rots on alfalfa and soybean in sterilized vermiculite and naturally infested field soil. The Streptomyces isolates tested significantly reduced root rot severity in alfalfa and soybean caused by P. medicaginis and P. sojae, respectively (P < 0.05). On alfalfa, isolates varied in their effect on plant disease severity, percentage dead plants, and plant biomass in the presence of the pathogen. The same eight isolates of Streptomyces were also tested for inhibitory activities against each other and against three strains of Bradyrhizobium japonicum and two strains of Sinorhizobium meliloti isolated from soybean and alfalfa, respectively. Streptomyces isolates clustered into two major compatibility groups: isolates within the same group were noninhibitory toward one another in vitro. The compatibility groups corresponded with groupings obtained based upon inhibition of B. japonicum and S. meliloti strains.     

The soybean-Phytophthora resistance locus <Emphasis Type="Italic">Rps1</Emphasis>-k encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive sequences

Background  

A series of Rps (resistance to P ytophthora s ojae) genes have been protecting soybean from the root and stem rot disease caused by the Oomycete pathogen, Phytophthora sojae. Five Rps genes were mapped to the Rps1 locus located near the 28 cM map position on molecular linkage group N of the composite genetic soybean map. Among these five genes, Rps1-k was introgressed from the cultivar, Kingwa. Rps1-k has been providing stable and broad-spectrum Phytophthora resistance in the major soybean-producing regions of the United States. Rps1-k has been mapped and isolated. More than one functional Rps1-k gene was identified from the Rps1-k locus. The clustering feature at the Rps1-k locus might have facilitated the expansion of Rps1-k gene numbers and the generation of new recognition specificities. The Rps1-k region was sequenced to understand the possible evolutionary steps that shaped the generation of Phytophthora resistance genes in soybean.     

Light and Electron Microscopy Studies on the Infection of a Wild‐type and Metalaxyl‐resistant Isolate of Phytophthora sojae in Soybean Hypocotyls

Resistance of soybean cultivars, depending on single dominant genes to Phytophthora sojae, may easily be overcome by emerging new virulent races. Light microscopy (LM) and electron microscopy (EM) were used to study the infection process of the wild‐type isolate Ps411 and metalaxyl‐resistant mutant Ps411‐M of P. sojae in hypocotyls of soybean seedlings grown from untreated and metalaxyl‐treated seeds. The isolate Ps411‐M of P. sojae exhibited a high degree of resistance to metalaxyl compared to Ps411. The pathogenic fitness of Ps411‐M in hypocotyls of soybean seedlings was lower compared to Ps411. LM observations showed distinct differences in the infection process of both isolates in hypocotyls of treated soybean seedlings. EM studies revealed differences in the prepenetration stage between Ps411 and Ps411‐M on hypocotyls grown from seeds treated with 0.02% metalaxyl until the whole seed surface coated. The number of infection sites was markedly reduced and few hyphae continued to spread. Numerous ultrastructural alterations in hyphae were observed in treated hypocotyls infected with Ps411, including pronounced thickening of hyphal cell walls and encasement of haustorium‐like bodies; electron‐dense material was deposited in host cell walls in contact with hyphal cells. Neither the prepenetration process nor penetration or spread of hyphae in the hypocotyls of the resistant isolate was affected in treated compared to non‐treated tissue. While in treated hypocotyls infected with the wild‐type isolate, host defence reactions were induced, no such reactions were detected in treated hypocotyls infected with the resistant isolate. Hypocotyls from metalaxyl‐treated seeds infected with the wild‐type isolate resembled an incompatible interaction, whereas during infection with the metalaxyl‐resistant mutant, the compatible interaction was not changed.     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

Identification and Candidate Gene Analysis of a Novel Phytophthora Resistance Gene Rps10 in a Chinese Soybean Cultivar

Resistance to Phytophthora sojae isolate PsMC1 was evaluated in 102 F_2∶3 families derived from a cross between the resistant soybean cultivar Wandou 15 and the susceptible cultivar Williams and genotyped using simple sequence repeat (SSR) markers. The segregation ratio of resistant, segregating, and susceptible phenotypes in the population suggested that the resistance in Wandou 15 was dominant and monogenic. Twenty-six polymorphic SSR markers were identified on soybean chromosome 17 (Molecular linkage group D2; MLG D2), which were linked to the resistance gene based on bulked segregation analysis (BSA). Markers Sattwd15-24/25 and Sattwd15-47 flanked the resistance gene at a distance of 0.5 cM and 0.8 cM, respectively. Two cosegregating markers, Sattwd15-28 and Sattwd15-32, were also screened in this region. This is the first Rps resistance gene mapped on chromosome 17, which is designated as Rps10. Eight putative genes were found in the mapped region between markers Sattwd15-24/25 and Sattwd15-47. Among them, two candidate genes encoding serine/threonine (Ser/Thr) protein kinases in Wandou 15 and Williams were identified and sequenced. And the differences in genomic sequence and the putative amino acid sequence, respectively, were identified within each candidate gene between Wandou 15 and Williams. This novel gene Rps10 and the linked markers should be useful in developing soybean cultivars with durable resistance to P. sojae.     

Virulence and RAPD data—A tool to study the evolutionary trends of Colletotrichum lindemuthianum virulences in the North Western Himalayan region of India

Abstract

Bean anthracnose pathogen (Colletotrichum lindemuthianum) known to display high pathogenic variability, also explains the existence of large number of races in Himachal Pradesh. An evolution model based on virulence data of 29 C. lindemuthianum races and RAPD patterns revealed the existence of four evolutionary groups (EG I – EG IV) in Himachal Pradesh, accommodating 12, 14, 2 and 1 races, respectively. Some races viz., 935, 643, 529, 647 and 613, opted more than two evolutionary routes and races like 598, 707, 935, 631, 639, 615, 115 and 119 harbouring more than six virulence genes may pose a threat to bean cultivation in this part of the world as they can break many resistance genes present in the locally grown beans. However, two exotic accessions G 2333 and AB 136 resistant to all the Indian pathotypes could be exploited as resistance donors in developing anthracnose resistant cultivars suitable for cultivation in this region.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.