A deep semantic network-based image segmentation of soybean rust pathogens.

Introduction: Asian soybean rust is a highly aggressive leaf-based disease triggered by the obligate biotrophic fungus Phakopsora pachyrhizi which can cause up to 80% yield loss in soybean. The precise image segmentation of fungus can characterize fungal phenotype transitions during growth and help to discover new medicines and agricultural biocides using large-scale phenotypic screens. Methods: The improved Mask R-CNN method is proposed to accomplish the segmentation of densely distributed, overlapping and intersecting microimages. First, Res2net is utilized to layer the residual connections in a single residual block to replace the backbone of the original Mask R-CNN, which is then combined with FPG to enhance the feature extraction capability of the network model. Secondly, the loss function is optimized and the CIoU loss function is adopted as the loss function for boundary box regression prediction, which accelerates the convergence speed of the model and meets the accurate classification of high-density spore images. Results: The experimental results show that the mAP for detection and segmentation, accuracy of the improved algorithm is improved by 6.4%, 12.3% and 2.2% respectively over the original Mask R-CNN algorithm. Discussion: This method is more suitable for the segmentation of fungi images and provide an effective tool for large-scale phenotypic screens of plant fungal pathogens.

Genetic Mapping of Seven Kinds of Locus for Resistance to Asian Soybean Rust.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most serious soybean (Glycine max) diseases in tropical and subtropical regions. To facilitate the development of resistant varieties using gene pyramiding, DNA markers closely linked to seven resistance genes, namely, Rpp1, Rpp1-b, Rpp2, Rpp3, Rpp4, Rpp5, and Rpp6, were identified. Linkage analysis of resistance-related traits and marker genotypes using 13 segregating populations of ASR resistance, including eight previously published by our group and five newly developed populations, identified the resistance loci with markers at intervals of less than 2.0 cM for all seven resistance genes. Inoculation was conducted of the same population with two P. pachyrhizi isolates of different virulence, and two resistant varieties, 'Kinoshita' and 'Shiranui,' previously thought to only harbor Rpp5, was found to also harbor Rpp3. Markers closely linked to the resistance loci identified in this study will be used for ASR-resistance breeding and the identification of the genes responsible for resistance.

Mapping of a soybean rust resistance in PI 594756 at the Rpp1 locus.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is the main disease affecting soybean in Brazil. This study aimed at investigating and mapping the resistance of the PI 594756 to P. pachyrhizi, by using Bulked Segregant Analysis (BSA). The PI 594756 and the susceptible PI 594891 were crossed and the resulting F2 and F2:3 populations (208 and 1770 plants, respectively) were tested against ASR. Also, these PIs and differential varieties were tested against a panel of monosporic isolates. Plants presenting tan lesions were classified as susceptible (S) while plants presenting reddish-brown (RB) lesions were classified as resistant. DNA bulks were genotyped with Infinium BeadChips and the genomic region identified was further analyzed in the F2 individuals with target GBS (tGBS). PI 594,56 presented a unique resistance profile compared to the differential varieties. The resistance was monogenic dominant; however, it was classified as incompletely dominant when quantitatively studied. Genetic and QTL mapping placed the PI 594756 gene between the genomic region located at 55,863,741 and 56,123,516 bp of chromosome 18. This position is slightly upstream mapping positions of Rpp1 (PI 200492) and Rpp1-b (PI 594538A). Finally, we performed a haplotype analysis in a whole genomic sequencing-SNP database composed of Brazilian historical germplasm and sources of Rpp genes. We found SNPs that successfully differentiated the new PI 594756 allele from Rpp1 and Rpp1-b sources. The haplotype identified can be used as a tool for marker-assisted selection (MAS). Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01358-4.

A genome-wide association study and genomic prediction for Phakopsora pachyrhizi resistance in soybean.

Soybean brown rust (SBR), caused by Phakopsora pachyrhizi, is a devastating fungal disease that threatens global soybean production. This study conducted a genome-wide association study (GWAS) with seven models on a panel of 3,082 soybean accessions to identify the markers associated with SBR resistance by 30,314 high quality single nucleotide polymorphism (SNPs). Then five genomic selection (GS) models, including Ridge regression best linear unbiased predictor (rrBLUP), Genomic best linear unbiased predictor (gBLUP), Bayesian least absolute shrinkage and selection operator (Bayesian LASSO), Random Forest (RF), and Support vector machines (SVM), were used to predict breeding values of SBR resistance using whole genome SNP sets and GWAS-based marker sets. Four SNPs, namely Gm18_57,223,391 (LOD = 2.69), Gm16_29,491,946 (LOD = 3.86), Gm06_45,035,185 (LOD = 4.74), and Gm18_51,994,200 (LOD = 3.60), were located near the reported P. pachyrhizi R genes, Rpp1, Rpp2, Rpp3, and Rpp4, respectively. Other significant SNPs, including Gm02_7,235,181 (LOD = 7.91), Gm02_7234594 (LOD = 7.61), Gm03_38,913,029 (LOD = 6.85), Gm04_46,003,059 (LOD = 6.03), Gm09_1,951,644 (LOD = 10.07), Gm10_39,142,024 (LOD = 7.12), Gm12_28,136,735 (LOD = 7.03), Gm13_16,350,701(LOD = 5.63), Gm14_6,185,611 (LOD = 5.51), and Gm19_44,734,953 (LOD = 6.02), were associated with abundant disease resistance genes, such as Glyma.02G084100, Glyma.03G175300, Glyma.04g189500, Glyma.09G023800, Glyma.12G160400, Glyma.13G064500, Glyma.14g073300, and Glyma.19G190200. The annotations of these genes included but not limited to: LRR class gene, cytochrome 450, cell wall structure, RCC1, NAC, ABC transporter, F-box domain, etc. The GWAS based markers showed more accuracies in genomic prediction than the whole genome SNPs, and Bayesian LASSO model was the ideal model in SBR resistance prediction with 44.5% ~ 60.4% accuracies. This study aids breeders in predicting selection accuracy of complex traits such as disease resistance and can shorten the soybean breeding cycle by the identified markers.

Phenotypic Evaluation of Soybean Genotypes for Their Reaction to a Mississippi Isolate of Phakopsora pachyrhizi Causing Soybean Rust.

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. and P. Syd. is one of the most important foliar diseases of soybean. SBR has the potential to cause major economic damage to global and U.S. soybean production. Analysis of reactions of soybean genotypes to P. pachyrhizi is an important step towards breeding for resistance to SBR. Fifty-four diverse soybean genotypes with both known and unknown Rpp resistance genes were tested for their reactions to a Mississippi P. pachyrhizi isolate. PI 567102B (Rpp6) had a near-immune reaction with the lowest disease severity score and no sporulation. Among seventeen genotypes with resistant or incomplete resistant reddish-brown (RB) reactions, eight are improved breeding lines that are available to researchers through material transfer agreements (MTAs). Thirty-six genotypes had the susceptible TAN reaction. Four soybean lines (RN06-32-1(7-b, GC 00138-29, G01-PR16, and GC 84051-9-1) had RB reactions and significantly lower SBR severity and sporulation than three of the six resistant checks, PI 230970 (Rpp2), PI 462312 (Rpp3), and PI 459025B (Rpp4). G01-PR16 is a publicly released germplasm. This research provides new information about reactions of different soybean genotypes to a midsouthern USA isolate of P. pachyrhizi and thereby aids in breeding for resistance to SBR.

Diverse environmental bacteria displaying activity against Phakopsora pachyrhizi, the cause of soybean rust.

The management of soybean rust (SBR) caused by the obligate fungus Phakopsora pachyrhizi mostly relies on the use of synthetic fungicides, especially in areas where the disease inflicts serious yield losses. The reliance on synthetic fungicides to manage this disease has resulted in resistance of P. pachyrhizi populations to most fungicides. In this study, bacteria isolated from diverse environments were evaluated for their biocontrol potential against P. pachyrhizi using soybean detached-leaf method and on-plant in the growth chamber, greenhouse, and field. Among 998 bacterial isolates evaluated using the detached-leaf method; 58% were isolated from plant-related materials, 27% from soil, 10% from insects, and 5% from other environments. Of the isolates screened, 73 were active (they had ⪖ 75% rust reduction) with an active rate of 7.3%. From the active isolates, 65 isolates were re-tested on-plant in the growth chamber for activity confirmation. In the confirmation test, 49 bacteria isolated from plant-related materials maintained their activity with a confirmation rate of 75%. The majority of bacteria with confirmed activity belonged to the taxonomic classes Bacilli and Gammaproteobacteria (70%). Active isolates were prioritized for greenhouse and field testing based on activity in the initial screen and confirmation test. Six bacterial isolates AFS000009 (Pseudomonas_E chlororaphis), AFS032321 (Bacillus subtilis), AFS042929 (Bacillus_C megaterium), AFS065981 (Bacillus_X simplex_A), AFS090698 (Bacillus_A thuringiensis_S), and AFS097295 (Bacillus_A toyonensis) were selected from those bacteria that maintained activity in the confirmation test and were evaluated in the greenhouse, and five among them were evaluated in the field. From the Alabama field evaluation, all bacterial isolates reduced rust infection as well as azoxystrobin (Quadris® at 0.3 L/ha) used as the fungicide control (P > 0.05). Moreover, the scanning electron micrographs demonstrated evidence of antagonistic activity of AFS000009 and AFS032321 against P. pachyrhizi urediniospores. Bacterial isolates that consistently showed activity comparable to that of azoxystrobin can be improved through fermentation and formulation optimization, developed, and deployed. These bacteria strains would provide a valuable alternative to the synthetic fungicides and could play a useful role in integrated disease management programs for this disease.

An NBS-LRR protein in the Rpp1 locus negates the dominance of Rpp1-mediated resistance against Phakopsora pachyrhizi in soybean.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

Understanding Phakopsora pachyrhizi in soybean: comprehensive insights, threats, and interventions from the Asian perspective.

Soybean (Glycine max L.) is an important crop in Asia, accounting for 17% of global soybean cultivation. However, this crop faces formidable challenges from the devastating foliar disease, Asian Soybean Rust (ASR), caused by Phakopsora pachyrhizi, a biotrophic fungus with a broad host range, causing substantial yield losses (10-100%) in Asia. This comprehensive review consolidates knowledge on ASR, encompassing its impact, historical perspectives, genetic diversity, epidemic drivers, early detection, risk assessment, and sustainable management strategies of ASR in the region. ASR has expanded globally from Asia, reaching Africa and Americas, driven by wind-dispersed urediniospores. Genetic diversity studies reveal the complexity of P. pachyrhizi, with distinct populations exhibiting varying virulence patterns. Factors affecting ASR epidemics in Asia include host susceptibility, landscape connectivity, climate, and environmental conditions. Understanding the interplay of these factors is essential for early intervention and control of ASR in soybean fields. Effectively managing ASR can exploit the utilization of diverse intervention strategies, encompassing disease forecasting, automated early detection, disease resistance, fungicide application, and biological control. A pivotal aspect of successful, sustainable disease management lies in reducing the ASR pathogen virulence and preventing it from developing fungicide resistance, while the highpoint of effectiveness in disease control is attained through a synergistic approach, integrating various strategies. In summary, this comprehensive review provides insights into multifaceted approaches that contribute to the development of sustainable and economically impactful soybean production in the face of the persistent threat of ASR in Asia.

Host-Induced Gene Silencing Using BPMV on Soybean to Study Genes in the Soybean Rust Fungus Phakopsora pachyrhizi.

To obtain direct evidence for the influence of an effector on the virulence or pathogenicity of a pathogen, it is necessary to knock out, knock down, or silence the respective gene. Since genetic transformation is not yet possible for rust fungi, silencing the gene is the only option. Posttranscriptional gene silencing uses RNAi. RNAi in plant pathogens can be accomplished by introducing dsRNA either by direct application of in vitro synthesized dsRNA or through positive-strand or double-strand RNA plant viruses. For studying effectors in Phakopsora pachyrhizi, we have implemented a host-induced silencing procedure based on virus-induced gene silencing using the bean pod mottle virus system. Here, procedures and interpretations of results are described and limitations of the system are discussed.

New Approaches to Manage Asian Soybean Rust (Phakopsora pachyrhizi) Using Trichoderma spp. or Their Antifungal Secondary Metabolites.

Attempts have been made to determine the in vitro and in planta suppressive potential of particular Trichoderma strains (T16 and T23) and their secondary metabolites (SMs) against Asian soybean rust (ASR) incited by Phakopsora pachyrhizi. Aside from the previously identified SMs 6-pentyl-α-pyrone (6PAP) and viridiofungin A (VFA), the chemical structures of harzianic acid (HA), iso-harzianic acid (iso-HA), and harzianolide (HZL) were characterized in this study. Our results indicate that exposure of urediospores to 200 ppm 6PAP completely inhibits germination. A slightly higher dosage (250 ppm) of HZL and VFA reduces germination by 53.7% and 44%, respectively. Germ tube elongation seems more sensitive to 6PAP than urediospore germination. On detached leaves, application of conidia of T16 and T23 results in 81.4% and 74.3% protection, respectively. Likewise, 200 ppm 6PAP recorded the highest ASR suppression (98%), followed by HZL (78%) and HA (69%). Treatment of undetached leaves with 6PAP, HA, or HZL reduces ASR severity by 84.2%, 65.8%, and 50.4%, respectively. Disease reduction on the next, untreated trifoliate by T23 (53%), T16 (41%), HZL (42%), and 6PAP (32%) suggests a translocation or systemic activity of the SMs and their producers. To our knowledge, this study provides the first proof for controlling ASR using antifungal SMs of Trichoderma. Our findings strongly recommend the integration of these innovative metabolites, particularly 6PAP and/or their producers in ASR management strategies.

Mapping QTLs Controlling Soybean Rust Disease Resistance in Chiang Mai 5, an Induced Mutant Cultivar.

Soybean rust (SBR) caused by the fungus Phakopsora pachyrhizi is an important folia disease of soybean (Glycine max). In this study, we identified QTLs controlling SBR in Chiang Mai 5 (CM5), an SBR-resistant cultivar developed by induced mutation breeding. A recombinant inbred line (RIL) population of 108 lines developed from a cross between Sukhothai 2 (SKT2, a susceptible cultivar) and CM5 was evaluated for SBR resistance under field conditions in Thailand. QTL analysis for the resistance in the RIL population identified a single QTL, qSBR18.1, for resistance. qSBR18.1 was mapped to a 212-kb region on chromosome 18 between simple sequence repeat markers Satt288 and sc21_3420 and accounted for 21.31-35.09% depending on the traits evaluated for resistance. The qSBR18.1 interval overlapped with genomic regions containing resistance to P. pachyrhizi 4 (Rpp4), a locus for SBR resistance. Three tightly linked genes, Glyma.18G226250, Glyma.18G226300, and Glyma.18G226500, each encoding leucine-rich repeat-containing protein, were identified as candidate genes for SBR resistance at the qSRB18.1. The qSBR18.1 would be useful for breeding of SBR resistance.

Covering Soybean Leaves With Cellulose Nanofiber Changes Leaf Surface Hydrophobicity and Confers Resistance Against Phakopsora pachyrhizi.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi, an obligate biotrophic fungal pathogen, is the most devastating soybean production disease worldwide. Currently, timely fungicide application is the only means to control ASR in the field. We investigated cellulose nanofiber (CNF) application on ASR disease management. CNF-treated leaves showed reduced lesion number after P. pachyrhizi inoculation compared to control leaves, indicating that covering soybean leaves with CNF confers P. pachyrhizi resistance. We also demonstrated that formation of P. pachyrhizi appressoria, and also gene expression related to these formations, such as chitin synthases (CHSs), were significantly suppressed in CNF-treated soybean leaves compared to control leaves. Moreover, contact angle measurement revealed that CNF converts soybean leaf surface properties from hydrophobic to hydrophilic. These results suggest that CNF can change soybean leaf surface hydrophobicity, conferring resistance against P. pachyrhizi, based on the reduced expression of CHSs, as well as reduced formation of pre-infection structures. This is the first study to investigate CNF application to control field disease.

Secondary Metabolites of Pseudomonas aeruginosa LV Strain Decrease Asian Soybean Rust Severity in Experimentally Infected Plants.

Asian Soybean Rust (ASR), a disease caused by Phakopsora pachyrhizi, causing yield losses up to 90%. The control is based on the fungicides which may generate resistant fungi. The activation of the plant defense system, should help on ASR control. In this study, secondary metabolites of Pseudomonas aeruginosa LV strain were applied on spore germination and the expression of defense genes in infected soybean plants. The F4A fraction and the pure metabolites were used. In vitro, 10 µg mL-1 of F4A reduced spore germination by 54%, while 100 µg mL-1 completely inhibited. Overexpression of phenylalanine ammonia lyase (PAL), O-methyltransferase (OMT) and pathogenesis related protein-2 (PR-2; glucanases) defense-related genes were detected 24 and 72 h after soybean sprouts were sprayed with an organocopper antimicrobial compound (OAC). Under greenhouse conditions, the best control was observed in plants treated with 60 µg mL-1 of PCA, which reduced ASR severity and lesion frequency by 75% and 43%, respectively. Plants sprayed with 2 and 20 µg mL-1 of F4A also decreased severity (41%) and lesion frequency (32%). The significant reduction in spore germination ASR in plant suggested that the strain of these metabolites are effective against P. pachyrhizi, and they can be used for ASR control.

Untargeted Metabolomics Analysis by UHPLC-MS/MS of Soybean Plant in a Compatible Response to Phakopsora pachyrhizi Infection.

Phakopsora pachyrhizi is a biotrophic fungus, causer of the disease Asian Soybean Rust, a severe crop disease of soybean and one that demands greater investment from producers. Thus, research efforts to control this disease are still needed. We investigated the expression of metabolites in soybean plants presenting a resistant genotype inoculated with P. pachyrhizi through the untargeted metabolomics approach. The analysis was performed in control and inoculated plants with P. pachyrhizi using UHPLC-MS/MS. Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA), was applied to the data analysis. PCA and PLS-DA resulted in a clear separation and classification of groups between control and inoculated plants. The metabolites were putative classified and identified using the Global Natural Products Social Molecular Networking platform in flavonoids, isoflavonoids, lipids, fatty acyls, terpenes, and carboxylic acids. Flavonoids and isoflavonoids were up-regulation, while terpenes were down-regulated in response to the soybean-P. pachyrhizi interaction. Our data provide insights into the potential role of some metabolites as flavonoids and isoflavonoids in the plant resistance to ASR. This information could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.

Identification and characterization of a new soybean promoter induced by Phakopsora pachyrhizi, the causal agent of Asian soybean rust.

BACKGROUND: Phakopsora pachyrhizi is a biotrophic fungal pathogen responsible for the Asian soybean rust disease causing important yield losses in tropical and subtropical soybean-producing countries. P. pachyrhizi triggers important transcriptional changes in soybean plants during infection, with several hundreds of genes being either up- or downregulated. RESULTS: Based on published transcriptomic data, we identified a predicted chitinase gene, referred to as GmCHIT1, that was upregulated in the first hours of infection. We first confirmed this early induction and showed that this gene was expressed as early as 8 h after P. pachyrhizi inoculation. To investigate the promoter of GmCHIT1, transgenic soybean plants expressing the green fluorescence protein (GFP) under the control of the GmCHIT1 promoter were generated. Following inoculation of these transgenic plants with P. pachyrhizi, GFP fluorescence was detected in a limited area located around appressoria, the fungal penetration structures. Fluorescence was also observed after mechanical wounding whereas no variation in fluorescence of pGmCHIT1:GFP transgenic plants was detected after a treatment with an ethylene precursor or a methyl jasmonate analogue. CONCLUSION: We identified a soybean chitinase promoter exhibiting an early induction by P. pachyrhizi located in the first infected soybean leaf cells. Our results on the induction of GmCHIT1 promoter by P. pachyrhizi contribute to the identification of a new pathogen inducible promoter in soybean and beyond to the development of a strategy for the Asian soybean rust disease control using biotechnological approaches.

Whole Genome Re-sequencing of Soybean Accession EC241780 Providing Genomic Landscape of Candidate Genes Involved in Rust Resistance.

BACKGROUND: In this study, whole genome re-sequencing of rust resistant soybean genotype EC241780 was performed to understand the genomic landscape involved in the resistance mechanism. METHODS: A total of 374 million raw reads were obtained with paired-end sequencing performed with Illumina HiSeq 2500 instrument, out of which 287.3 million high quality reads were mapped to Williams 82 reference genome. Comparative sequence analysis of EC241780 with rust susceptible cultivars Williams 82 and JS 335 was performed to identify sequence variation and to prioritise the candidate genes. RESULTS: Comparative analysis indicates that genotype EC241780 has high sequence similarity with rust resistant genotype PI 200492 and the resistance in EC241780 is conferred by the Rpp1 locus. Based on the sequence variations and functional annotations, three genes Glyma18G51715, Glyma18G51741 and Glyma18G51765 encoding for NBS-LRR family protein were identified as the most prominent candidate for Rpp1 locus. CONCLUSION: The study provides insights of genome-wide sequence variation more particularly at Rpp1 loci which will help to develop rust resistant soybean cultivars through efficient exploration of the genomic resource.

Performance and Profitability of Rain-Based Thresholds for Timing Fungicide Applications in Soybean Rust Control.

Soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi, is the most damaging disease of soybean in Brazil. Effective management is achieved by means of calendar-timed sprays of fungicide mixtures, which do not explicitly consider weather-associated disease risk. Two rain-based action thresholds of disease severity values (DSV50 and DSV80) were proposed and compared with two leaf wetness duration-temperature thresholds of daily values of infection probability (DVIP6 and DVIP9) and with a calendar program, with regard to performance and profitability. An unsprayed check treatment plot was included for calculating relative control. Disease severity and yield data were obtained from 29 experiments conducted at six sites across four states in Brazil during the 2012-13, 2014-15, and 2015-16 growing seasons, which represented different growing regions and climatic conditions. The less conservative rainfall action threshold (DSV80) resulted in fewer fungicide sprays compared with the other treatments, and the more conservative one (DSV50) resulted in fewer sprays than the DVIP thresholds. Yield was generally higher with the increase in spray number, but the economic analysis showed no significant differences in the risk of not offsetting the costs of fungicide sprays regardless of the system. Therefore, based on the simplicity and the profitability of the rain-based model, the system is a good candidate for incorporating into the management of SBR in soybean production fields in Brazil.

Field Resistance to Phakopsora pachyrhizi and Colletotrichum truncatum of Transgenic Soybean Expressing the NmDef02 Plant Defensin Gene.

Fungal diseases lead to significant losses in soybean yields and a decline in seed quality; such is the case of the Asian soybean rust and anthracnose caused by Phakopsora pachyrhizi and Colletotrichum truncatum, respectively. Currently, the development of transgenic plants carrying antifungal defensins offers an alternative for plant protection against pathogens. This paper shows the production of transgenic soybean plants expressing the NmDef02 defensin gene using the biolistic delivery system, in an attempt to improve resistance against diseases and reduce the need for chemicals. Transgenic lines were assessed in field conditions under the natural infections of P. pachyrhizi and C. truncatum. The constitutive expression of the NmDef02 gene in transgenic soybean plants was shown to enhance resistance against these important plant pathogens. The quantification of the P. pachyrhizi biomass in infected soybean leaves revealed significant differences between transgenic lines and the non-transgenic control. In certain transgenic lines there was a strong reduction of fungal biomass, revealing a less severe disease. Integration and expression of the transgenes were confirmed by PCR, Southern blot, and qRT-PCR, where the Def1 line showed a higher relative expression of defensin. It was also found that the expression of the NmDef02 defensin gene in plants of the Def1 line did not have a negative effect on the nodulation induced by Bradyrhizobium japonicum. These results indicate that transgenic soybean plants expressing the NmDef02 defensin gene have a substantially enhanced resistance to economically important diseases, providing a sound environmental approach for decreasing yield losses and lowering the burden of chemicals in agriculture.

Reduction of Phakopsora pachyrhizi infection on soybean through host- and spray-induced gene silencing.

Asian soybean rust (ASR), caused by the obligate fungal pathogen Phakopsora pachyrhizi, often leads to significant yield losses and can only be managed through fungicide applications currently. In the present study, eight urediniospore germination or appressorium formation induced P. pachyrhizi genes were investigated for their feasibility to suppress ASR through a bean pod mottle virus (BPMV)-based host-induced gene silencing (HIGS) strategy. Soybean plants expressing three of these modified BPMV vectors suppressed the expression of their corresponding target gene by 45%-80%, fungal biomass accumulation by 58%-80%, and significantly reduced ASR symptom development in soybean leaves after the plants were inoculated with P. pachyrhizi, demonstrating that HIGS can be used to manage ASR. In addition, when the in vitro synthesized double-stranded RNAs (dsRNAs) for three of the genes encoding an acetyl-CoA acyltransferase, a 40S ribosomal protein S16, and glycine cleavage system H protein were sprayed directly onto detached soybean leaves prior to P. pachyrhizi inoculation, they also resulted in an average of over 73% reduction of pustule numbers and 75% reduction in P. pachyrhizi biomass accumulation on the detached leaves compared to the controls. To the best of our knowledge, this is the first report of suppressing P. pachyrhizi infection in soybean through both HIGS and spray-induced gene silencing. It was demonstrated that either HIGS constructs targeting P. pachyrhizi genes or direct dsRNA spray application could be an effective strategy for reducing ASR development on soybean.

Reactions of Soybean Germplasm Accessions to Six Phakopsora pachyrhizi Isolates from the United States.

Soybean rust, caused by Phakopsora pachyrhizi Syd. & P. Syd., is one of the most economically important foliar diseases of soybean. Resistant cultivars could reduce yield losses and management costs but considerable pathogenic diversity exists among populations of the fungus; thus, resistance to a range of pathotypes is essential. Seedling and detached-leaf assays were conducted to characterize the resistance of 55 soybean plant introductions (PIs) to six purified isolates of P. pachyrhizi originating from the southern United States. In the greenhouse resistance assays, the differentials Hyuuga (PI 506764) and PI 471904 and accessions PI 224268, PI 567025A, PI 567039, PI 567046A, and DT 2000 (PI 635999) were resistant to all six isolates, including Florida isolates from 2011 and 2012 that were able to defeat resistance conditioned by the Rpp1 through Rpp4 genes. Twenty-six other PIs were resistant to four or five of the six isolates. In the detached-leaf assays, eight accessions developed reddish-brown reactions to all six isolates, with an average of only 0.23 to 0.55 uredinia/lesion. These included Hyuuga, DT 2000, two differentials with a resistance allele at the Rpp5 locus, and accessions PI 224268, PI 423960B, PI 567025A, and PI 567046A. Many of the resistant accessions have subsequently been reported to have a resistance allele at the Rpp3 locus, and two others have resistance genes at the Rpp4 or Rpp6 locus. This study provided new information about resistance reaction phenotypes that can be useful for understanding mechanisms of resistance, which Rpp genes and alleles could be combined to obtain broader and more durable rust resistance in soybean cultivars, and pathotype diversity among the six isolates used.