Soybean Disease Research Articles

ОЦЕНКА УСТОЙЧИВОСТИ СОРТОВ СОИ К БАКТЕРИАЛЬНЫМ БОЛЕЗНЯМ НА ИСКУССТВЕННОМ ИНФЕКЦИОННОМ ФОНЕ

Bacterial blight (P. savastanoi pv. glycinea (Psg)) is one of the main bacterial diseases reducing the profitability of soybean cultivation. Another soybean disease recently discovered in Russia is rustybrown bacterial spot (and wilting) (C. flaccumfaciens pv. flaccumfaciens). The harm caused by these diseases, the emergence of antibiotic-resistant strains of bacterial phytopathogens and the imperfection of available protection necessitate the search for alternative strategies, one of which is the use of resistant or low-strucking cultivars. In this work, the optimal method of artificial inoculation of plants was determined and 47 domestic soybean cultivars were screened for susceptibility to different strains of the two pathogens. It is shown that the optimal method of inoculation is cutting with scissors soaked in a suspension of bacteria with a concentration of 108 CFU/mL, which produces typical disease symptoms with a maximum damage area in 10 days after inoculation. The least affected cultivar was Soer 4, and the most susceptible cultivars were Nordica and Osmon. An atypical hypersensitivity reaction was noted in the form of abortion of a triple leaf with a petiole during Psg inoculation in cultuvars Nordica and Opus and cultivar Maxus after Psg and Cff inoculation

Causal variant loci and protein-coding genes for soybean bacterial pustule resistance in the flowering stage

Bacterial pustule is an important soybean disease caused by Xanthomonas citri pv. glycines, but information about genetic resistance to this pathogen is scarce. This study aimed to investigate soybean genetic resistance to bacterial pustule in the flowering stage through association analysis, characterization of candidate SNP markers, and identification of protein-coding genes potentially regulating defense processes. Therefore, 109 soybean cultivars genotyped with a 6k Illumina platform were assessed for disease severity. A genome-wide analysis revealed a total of 13 SNPs significantly associated. Through protein annotation, we identified three markers located inside the coding regions of uncharacterized protein LOC100779077, histone-lysine N-methyltransferase SUVR4, and ABC-transporter B family member-9. Nucleotide polymorphism on the first two of these markers produces a non-synonymous polymorphism with polarity shift from hydrophobic to polar amino acid. It is convenient to prioritize these three candidate markers for validation procedures with the purpose of using them in marker-assisted soybean breeding programs.

Mapping Major Disease Resistance Genes in Soybean by Genome-Wide Association Studies.

Soybean is one of the most valuable agricultural crops in the world. Besides, this legume is constantly attacked by a wide range of pathogens (fungi, bacteria, viruses, and nematodes) compromising yield and increasing production costs. One of the major disease management strategies is the genetic resistance provided by single genes and quantitative trait loci (QTL). Identifying the genomic regions underlying the resistance against these pathogens on soybean is one of the first steps performed by molecular breeders. In the past, genetic mapping studies have been widely used to discover these genomic regions. However, over the last decade, advances in next-generation sequencing technologies and their subsequent cost decreasing led to the development of cost-effective approaches to high-throughput genotyping. Thus, genome-wide association studies applying thousands of SNPs in large sets composed of diverse soybean accessions have been successfully done. In this chapter, a comprehensive review of the majority of GWAS for soybean diseases published since this approach was developed is provided. Important diseases caused by Heterodera glycines, Phytophthora sojae, and Sclerotinia sclerotiorum have been the focus of the several GWAS. However, other bacterial and fungi diseases also have been targets of GWAS. As such, this GWAS summary can serve as a guide for future studies of these diseases. The protocol begins by describing several considerations about the pathogens and bringing different procedures of molecular characterization of them. Advice to choose the best isolate/race to maximize the discovery of multiple R genes or to directly map an effective R gene is provided. A summary of protocols, methods, and tools to phenotyping the soybean panel is given to several diseases. We also give details of options of DNA extraction protocols and genotyping methods, and we describe parameters of SNP quality to soybean data. Websites and their online tools to obtain genotypic and phenotypic data for thousands of soybean accessions are highlighted. Finally, we report several tricks and tips in Subheading 4, especially related to composing the soybean panel as well as generating and analyzing the phenotype data. We hope this protocol will be helpful to achieve GWAS success in identifying resistance genes on soybean.

First Report of Soybean Seedling Disease caused by Rhizoctonia solani AG-7 in Taiwan.

From August to November 2020, reduced emergence and damping-off of soybean seedlings were observed in two fields (Benzhou and Wandan) in Taiwan. Disease incidence was approximately 40% in Benzhou by field scouting. The roots of damping-off seedlings were brown. Affected seedlings could be easily pulled out from the soil and the lesions on the roots/stem were generally dry and sunken. These symptoms suggested the possibility of Rhizoctonia infection. Soil surrounding symptomatic seedlings were collected to bait the potential pathogen and symptomatic plants were used for pathogen isolation. The diseased tissues were washed with tap water and surface-disinfected with 1% bleach before placing on the Dexon selection medium at 26°C for 2 days (Ko and Hora 1971). Hyphae were transferred to potato dextrose agar (PDA), and a brown colony with brown and irregular-shaped sclerotia grew from 90 out of 99 isolates. The hyphae exhibited typical characteristics of Rhizoctonia solani, including a constriction and a septum near the end of branching hyphae (Ajayi-Oyetunde and Bradely, 2018). Two isolates from Benzhou and two isolates from Wandan were tested for their pathogenicity, and eight surface-disinfected seeds were distributed evenly on the water agar plates covered by 2-day-old mycelia at 25°C in dark for 7 days. All isolates caused cotyledon rot and reduced germination. To verify their pathogenicity in pots, double-sterilized sorghum seeds were inoculated with two strains and incubated at 25°C for 2 weeks to be used as fungal inoculum (Ajayi-Oyetunde and Bradely, 2017). A layer of 15 ml of fungal inoculum was placed 5 cm beneath the soil surface in pots. Four soybean seeds were planted approximately 3 cm above the inoculum in each pot. After two weeks, reddish lesions on the hypocotyls or taproots of all seedlings in the inoculated pots were observed, while seedlings in the control pots inoculated with sterile sorghum seeds remained healthy. The pathogen was re-isolated from lesions and had identical morphology to the original isolates. To characterize the fungal identity, the internal transcribed spacer (ITS) was sequenced using the primers ITS1/ITS4 (Sharon et al., 2006). Using BLASTN in the NCBI database, the sequence (GenBank no. MW410857 and MW410858) showed 100% (639/639 bp) similarity to KF907734 and 99.83% (635/636 bp) similarity to AF354099, both belong to R. solani anastomosis group 7 (AG-7) (Hua et al. 2014; Gonzalez et al. 2001). Phylogenetic analysis comparing sequences with different AGs (Ajayi-Oyetunde and Bradely, 2017) grouped our isolates within the AG-7 clade with a 100% bootstrap confidence. In the anastomosis test, an incompatible zonation and unequal mycelial growth rates were observed when AG-7 isolates were paired with an AG-1 IA isolate. On the other hand, the compatible tuft reaction was observed when two AG-7 isolates were paired, and the compatible merge reaction was observed in the self-pairing tests (Macnish et al. 1997). Accordingly, the molecular and morphological characterizations confirmed the causal pathogen as R. solani AG-7. R. solani AG-7 was first reported on radishes in Japan (Homma et al., 1983), first found on carnation in Taiwan (Lo et al., 1990), and in field soils of various crops but not soybean (Chuang, 1997). It was suggested that Rhizoctonia diseases of soybean may be present in Taiwan, but molecular confirmation was lacking (Anonymus, 1979). As R. solani AG-7 causes diseases of soybean in the US and Japan (Baird et al., 1996), the importance of AG-7 as an endemic pathogen of soybean in Taiwan should be recognized and its prevalence determined as a first step to managing this disease.

Influence of biostimulants and biostimulating complexes on the growth and development of soybeans in the Western Forest-steppe of Ukraine

Goal. The choice of biocomplexes for providing crop’s maximal potential in the terms of organic farming.
 Methods. The biological preparations, stimulants, their combinations and systematic complexes were the objects of researches. The biotechnological researches methods used during the work. The preparations efficiency was determined by different consumption rates against fungi diseases. The records conducted as per approved techniques with experimental methods usage in phytopathology and plant protection.
 Results. The complex usage: Biomag Soybean 4.0 l/t + Biophosphoryn 2.0 l/t + PhytoDoctor1.0 l/t + Urozhay Start 0.1 l/t (the increased rate Biophosphoryn) showed the best results during the analysis of biological complexes for treating soybean seeds and their phytosynthetic abilities. The conducted analysis of different preparations showed that soybean vegetation indexes and plants resistance to diseases had clearly formed in dependence upon the combination of researched preparations. The best result of first researches seria have received by the complex: Biomag Soybean + extender (2.0 + 1.0 l/t) + Biophosphoryn (1.0 l/t) + Phytodoctor(1.0 l/t); treating at growing seed Gaubsin FORTE (1,5 l/ha);Uroshay Bor (1.5 l/ha), Aminostym (1.0 l/ha), Actarophyt (0,4—0,6 l/ha), FitoNiss (0.5 l/ ha), Urozhay Co-Mo(0.25 l/ha), Urozhay Co-Mo (0.35 l/ha). The highest indexes were received during the seeds complex usage: Biomag Soybean + Extender (2.0 + 1.0 l/t) + Biophosphorin (1.0 l/t) + Phytodoctor (1.0 l/t), treating at the growing: Gaubsin FORTE (1.5 l/ha); Urozhay Bor (1.5 l/ha), Biomag (11.0 l/ha); Actarophit (0.4—0.6 l/ha), BioMag (0,5 l/ ha), Urozhay Co-Mo (0.35 l/ha).
 Conclusions. Increasing the yield and reducing the manifestation of the complex of fungal diseases of soybeans can be achieved through the correct selection of a system of biofungicides and growth stimulants without the use of drugs of chemical origin.

Feasibility of Hybrid PSO-ANN Model for Identifying Soybean Diseases

Soybean disease has become one of vital factors restricting the sustainable development of high-yield and high-quality soybean industry. A hybrid artificial neural network (ANN) model optimized via particle swarm optimization (PSO) algorithm, which is denoted as PSO-ANN, is proposed in this paper for soybean diseases identification based on categorical feature inputs. Augmentation dataset is created via Synthetic minority over-sampling technique (SMOTE) to deal with quantitative insufficiency and categorical unbalance of the dataset. PSO algorithm is used to optimize the parameters in ANN, including the activation function, the number of hidden layers, the number of neurons in each hidden layer and the optimizer. In the end, ANN model with 2 hidden layers, 63 and 61 neurons in hidden layers respectively, Relu activation function and Adam optimizer yields the best overall test accuracy of 92.00%, compared with traditional machine learning methods. PSO-ANN shows superiority on various evaluation metrics, which may have great potential in crop diseases control for modern agriculture.

Smarter Pest Identification Technology (SPIDTECH): a Mobile Application for Digital Identification and Remote Monitoring of Insect Pests and Diseases of Major Crops in the Philippines

Correct identification and proper monitoring are vital components of integrated pest and disease management. The Android application – Smarter Pest Identification Technology (SPIDTECH) – was developed for the digital identification and remote monitoring of insect pests and diseases of rice, corn, coffee, cacao, banana, coconut, sugarcane, tomato, and soybean in the Philippines. SPIDTECH has three main features: the digital identification of insect pests and diseases using a smartphone camera; the digital library that contains images, identification signs, life stages, management practices, and other pertinent information about a pest or disease; and the remote monitoring that enables real-time mapping of reports through user-contributed images and GPS points. The application can identify 71 insects and 63 diseases, while the library features a collection of 104 insects and 89 diseases available in English and Filipino. The identification uses a pretrained MobileNetV2, a convolutional neural network design by Google for mobile devices. The models were retrained using the pest and disease image dataset collected specifically for this study and logging 55.7–73.3% accuracy. If connected to the internet, devices send image data from the field that are validated and used for model retraining. SPIDTECH recorded more than 7,400 device downloads from March 2019–July 2021 and received 4.8 out of 5 user review ratings. Based on the user-provided registration data in the same period, it has more than 5,600 registered users from 81 provinces with an average user acquisition rate of 266 new users per month. With the current features deployed, the application received more than 8,000 identification requests for different crops. The application has gathered a significant number of users necessary to evaluate the feasibility of the application in assisting in pest and disease identification and remote monitoring for the major crops in the Philippines.

A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.

Physics-informed deep learning characterizes morphodynamics of Asian soybean rust disease

Medicines and agricultural biocides are often discovered using large phenotypic screens across hundreds of compounds, where visible effects of whole organisms are compared to gauge efficacy and possible modes of action. However, such analysis is often limited to human-defined and static features. Here, we introduce a novel framework that can characterize shape changes (morphodynamics) for cell-drug interactions directly from images, and use it to interpret perturbed development of Phakopsora pachyrhizi, the Asian soybean rust crop pathogen. We describe population development over a 2D space of shapes (morphospace) using two models with condition-dependent parameters: a top-down Fokker-Planck model of diffusive development over Waddington-type landscapes, and a bottom-up model of tip growth. We discover a variety of landscapes, describing phenotype transitions during growth, and identify possible perturbations in the tip growth machinery that cause this variation. This demonstrates a widely-applicable integration of unsupervised learning and biophysical modeling.

WAASB index revealed stable resistance sources for soybean anthracnose in India

AbstractAnthracnose caused byColletotrichum truncatumis a major soybean disease in India. Genetic resistance is the viable option to combat yield losses due to this disease. In the current study, 19 soybean genotypes were evaluated for anthracnose disease resistance at five locations (Medziphema, Palampur, Dharwad, Jabalpur and Indore) for three consecutive years (2017–2019) to identify stable and superior genotypes as resistant sources and to elucidate genotype (G) × environment (E) interactions. Genotype effect, environment effect and G × E interactions were found significant (P< 0.001) where G × E interactions contributed highest (42.44) to the total variation followed by environment (29.71) and genotype (18.84). Through Weighted Average of Absolute Scores (WAASB) stability analysis, PS 1611 (WAASB score = 0.33) was found to be most stable and through WAASBY superiority analysis NRC 128 (WAASBY score = 94.31) and PS 1611 (WAASBY score = 89.43) were found to be superior for mean performance and stability. These two genotypes could be candidate parents for breeding for durable and stable anthracnose resistance. Through principal component analysis, disease score was found to be positively associated with relative humidity, wind speed at 2 m above ground level, effect of temperature on radiation use efficiency and global solar radiation based on latitude and Julian day. Among the five locations, Indore was found to be highly discriminative with the highest mean disease incidence and could differentiate anthracnose-resistant and susceptible genotypes effectively, therefore can be considered an ideal location for breeding for field resistance against anthracnose disease.

Diversity of Bacillus spp. from soybean phyllosphere as potential antagonist agents for Xanthomonas axonopodis pv. glycines causal of pustule disease

Abstract. Nurcahyanti SD, Wahyuni WS, Masnilah R, Nurdika AAH. 2021. Diversity of Bacillus spp. from soybean phyllosphere as potential antagonist agents for Xanthomonas axonopodis pv. glycines causal of pustule disease. Biodiversitas 22: 5003-5011. Pustule disease caused by Xanthomonas axonopodis pv. glycines (Xag) is an important disease in soybean. Bacteria from soybean phyllosphere is one of the potential biological agents against this disease. This is because the microorganisms have similarity. This study aimed to determine the diversity and species of bacteria from the soybean phyllosphere that have potential as biological agents. The research was carried out by morphological observation, physiological - biochemical testing, and molecularly with BOX AIR primer. Molecular identification was carried out by amplifying the 16S-rRNA gene with 27F and 1492R primers. The results showed that 11 isolates could inhibit Xag in vitro which showed morphological, biochemical, and molecular diversity. These bacteria were identified as Bacillus spp. which consisted of 4 groups, namely Bacillus siamensis, B. subtilis. B. amyloliquifaction and B. velezensis. The diversity of phyllosphere bacteria allows them to be used as biological agents because they do not inhibit each other and have diverse living abilities in various environmental conditions.

Evaluation of Scopoletin from Penicillium janthinellum Snef1650 for the Control of Heterodera glycines in Soybean.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is responsible for causing a major soybean disease globally. The fungal strain Penicillium janthinellum Snef1650 was evaluated against H. glycines. However, the effective determinants of the P. janthinellum strain are unknown. By performing pot experiments, a functioning compound was isolated from P. janthinellum Snef1650 through organic solvent extraction, semi-preparative HPLC, Sephadex LH-20 column chromatography, and silica gel column chromatography, and the isolated compound was identified to be scopoletin through 1H NMR, 13C NMR, and HPLC–MS. The pot experiments indicated that the treatment of soybean seeds with scopoletin drastically reduced the SCN population. The field experiments performed in 2017 and 2018 revealed that scopoletin decreased over 43.7% juveniles in the roots and over 61.55% cysts in the soil. Scopoletin treatment also promoted soybean growth and improved its yield, with an increase in plot yield by >5.33%. Scopoletin obtained from P. janthinellum Snef1650 could be used as an anti-H. glycines biocontrol agent.

Crop-weather based relation and severity prediction of aerial blight incited by Rhizoctonia solani Kuhn in soybean


 Crop-weather based relation and other aspects of aerial blight incited by Rhizoctoniasolani Kuhn were investigated on two mega varieties (JS 335 and JS 97-52, now susceptible) under central Indian conditions during 2017, 2018 and 2019. It was found that aerial severity and sclerotial formation on affected leaves were varied significantly in all three season, and progress of disease was rapid between 63–84 days old crop {full pod (R4) to maturity initiation (R7) stage}.Increasing crop age was also significantly positively correlated with increasing severity (0.732*) and sclerotial formation (0.981**). Mean relative humidity and maximum temperature of current as well as previous week were found to be positively and negatively correlated with increasing severity of aerial blight, respectively. A regression based model with three explanatory variables (Mean RH, Rainfall and Minimum temperature) of current week was found to be most significant in prediction of disease severity (R2=0.946) of aerial blight. Whereas previous week weather variables i.e. rainy days and rainfall were also accounted 71.7 per cent variability (R2 = 0.717) in disease severity. Overall, weekly average maximum temp (27 to 30o C) and mean RH (80 to 90 per cent) in current week coupled with more rainfall and rainy days in previous week were found to be most conducive field condition for rapid progress of aerial blight disease in soybean.

Agronomic and efficacy evaluations of oxathiapiprolin as a soybean seed treatment

AbstractPhytophthora root and stem rot is a devastating disease of soybean [Glycine max (L.) Merr.] that occurs annually in North America. Plant death and severe yield loss can transpire when conditions are favorable for pathogen development. The first objective was to determine the efficacy of oxathiapiprolin as a soybean seed treatment using laboratory inoculum layer assays with different races of Phytophthora sojae. The second objective was to evaluate oxathiapiprolin seed treatments in wide area agronomic field testing using unmanned aerial vehicle (UAV) image‐to‐data analysis to quantify emergence, vigor, and greenness traits. The final objective was to examine the yield potential of seed treatments with oxathiapiprolin. Using inoculum layer efficacy testing in a growth chamber with P. sojae races 4, 7, or 31, seed treated with oxathiapiprolin had significantly higher visual root growth scores compared to non‐treated seed. Across 115 field locations from 2018 to 2020, UAV remote sensing image analysis at the V2 growth stage indicated oxathiapiprolin in combination with other fungicides had significantly better emergence, more vigor, and more intense greenness compared to non‐treated controls. Entries treated with oxathiapiprolin in combination with other fungicides had an average +161 kg ha–1 yield difference compared to non‐treated entries from 2018 to 2020. Oxathiapiprolin as a seed treatment is an effective management tool for soybean producers, providing efficacy against P. sojae, and improving emergence, vigor, greenness, and final yield.

Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes.

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Assessment of Quinone Outside Inhibitor Sensitivity and Frogeye Leaf Spot Race of Cercospora sojina in Georgia Soybean.

Frogeye leaf spot (FLS), caused by the fungal pathogen Cercospora sojina K. Hara, is a foliar disease of soybean (Glycine max L. [Merr.]) responsible for yield reductions throughout the major soybean-producing regions of the world. In the United States, management of FLS relies heavily on the use of resistant cultivars and in-season fungicide applications, specifically within the class of quinone outside inhibitors (QoIs), which has resulted in the development of fungicide resistance in many states. In 2018 and 2019, 80 isolates of C. sojina were collected from six counties in Georgia and screened for QoI fungicide resistance using molecular and invitro assays, with resistant isolates being confirmed from three counties. Additionally, 50 isolates, including a "baseline isolate" with no prior fungicide exposure, were used to determine the percent reduction of mycelial growth to two fungicides, azoxystrobin and pyraclostrobin, at six concentrations: 0.0001, 0.001, 0.01, 0.1, 1, and 10 μg ml-1. Mycelial growth observed for resistant isolates varied significantly from both sensitive isolates and baseline isolate for azoxystrobin concentrations of 10, 1, 0.1, and 0.01 μg ml-1 and for pyraclostrobin concentrations of 10, 1, 0.1, 0.01, and 0.001 μg ml-1. Moreover, 40 isolates were used to evaluate pathogen race on six soybean differential cultivars by assessing susceptible or resistant reactions. Isolate reactions suggested 12 races of C. sojina present in Georgia, 4 of which have not been previously described. Species richness indicators (rarefaction and abundance-based coverage estimators) indicated that within-county C. sojina race numbers were undersampled in this study, suggesting the potential for the presence of either additional undescribed races or known but unaccounted for races in Georgia. However, no isolates were pathogenic on 'Davis', a differential cultivar carrying the Rcs3 resistance allele, suggesting that the gene is still an effective source of resistance in Georgia.

First report of soybean stem blight caused by Diaporthe phaseolorum in Sichuan province, China.

In September 2020, a disease resembling stem blight on soybean was found in Chengdu city, Sichuan province, southwestern of China. Symptoms began as a brown lesion on the stems, usually at the nodes, then lesions expanded, darkened, and even girdled the stems, causing wilt of the above stems (Figure 1A). In three 0.33-ha fields, a total of 300 soybean plants (20 plants/site × 5 sites/filed × 3 fields) were investigated, and 3% of the plants showed the disease symptoms. The symptoms were consistent with those previously reported for stem canker and stem blight on soybean caused by Diaporthe complex (Cui et al. 2009; Mena et al. 2019; Santos et al. 2011). The tissues of symptomatic soybean stems were rinsed by water, disinfected by submerging them in 75% ethanol for 30 s and in 2% sodium hypochlorite solution for 2 min, then followed by washing with sterile distilled water. Small diseased tissue fragments were placed on selective potato dextrose agar (PDA) containing rifampicin and ampicillin (both 50 mg/μl). Plates were sealed and incubated at 26°C for two days. Developing mycelia of isolates were transferred to fresh PDA and purified by single hyphal tip. For the five obtained isolates (Figure 1B), five markers, including the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, parts of the translation elongation factor 1-α (TEF1), part of the histone H3 (HIS) gene, the calmodulin gene (CAL), and the beta-tubulin gene (TUB), were amplified using the established primers ITS4/ITS5 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), CYLH3F (Crous et al. 2006) and H3-1b (Glass and Donaldson 1995), CAL228F/CAL737R (Carbone and Kohn 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995), respectively, and sequenced (GenBank IDs: MW595761-MW595780 and MW624472-MW624476). Phylogenetic trees were constructed based on concatenated sequences of the five markers using the maximum-likelihood (ML) method in MEGA-5.2.2. Based the results of morphological (Figure 1C-E) and phylogenetic analysis (Figure 2), the five isolates were all identified as D. phaseolorum. Pathogenicity tests for the isolates were conducted on 7-day-old soybean seedlings (cv. Shangdou 1310) using a hypocotyl slit inoculation method. At the stem 2-cm below cotyledon, a 6-mm long slit was cut with a sterile scalpel, and placed inside with a 3 mm × 3 mm PDA plug with or without mycelia of pathogen. Ten plants were assayed for each treatment, and the plants were maintained in greenhouse at 26°C, with humidity higher than 90% for the first two days. The assay was repeated at least three times. Typical brown lesions on the stems were observed four days after inoculation (Figure 1F), even 20% treated plants died. The D. phaseolorum was reisolated from these stem lesions. No disease symptom was observed on control plants (Figure 1G). Thus D. phaseolorum was the pathogen causing the soybean stem blight in field. To our knowledge, this is the first report of D. phaseolorum causing this disease in Sichuan province, China. The result may provide useful information for soybean disease control in this region of China. The authors declare no conflict of interest. Funding: China Agriculture Research System (CARS-004-PS14).

Cultural, Morphological Characterization and Aggressiveness of Alternaria alternata Causing Leaf Spot of Soybean

Background: Alternaria leaf spot caused by Alternaria alternata is one of the most important and destructive disease of soybean causing severe yield loss in all soybean growing areas of southern and eastern part of Rajasthan. Successful management of Alternaria leaf spot is mainly dependent on accurate and efficient detection of pathogen, amount of genetic and pathogenic variability present in pathogen population. The main reason for frequent “breakdown” of effective resistance is the variability that exists in the pathogen population, which necessitates a continual replacement of cultivars due to disease susceptibility. Methods: The twelve fungal isolates randomly were collected from six districts of major soybean growing part of Rajasthan i.e., Udaipur, Chittorgarh, Pratapgarh, Kota, Baran and Jhalawar. The culture was purified single spore techniques. These were then further compared among each other for any variations in cultural characters, colour of colonies, Growth rate, conidial morphology and pathogenic variability. Result: Twelve different isolates of A. alternata were obtained in pure culture and characterized for cultural, morphological variation and aggressiveness of this fungus varied in their cultural characters, colour of colonies, growth rate of isolates, conidial morphology and isolates also exhibited variations in incubation period, latent period, number and size of lesions were produced.

The еffect of fungicides on the development of diseases and soybean yield in the Forest steppe of Ukraine

Goal. To study the effectiveness of modern fungicides against the most common diseases of soybeans during the growing season and their effect on crop productivity in the Forest-steppe of Ukraine.
 Methods. Field, phytopathological, statistical. The experiments were carried out in the farms of the Kyiv region, belonging to the Forest-steppe zone of Ukraine. Spraying of soybean crops was carried out twice during the growing season in the phases of budding-beginning of flowering (51—55) and the formation of beans (71—75). Determined the development of diseases, the effectiveness of fungicides, crop yield.
 Results. The most common diseases of soybeans in the forest-steppe zone of Ukraine have been identified: Alternaria, downy mildew, Fusarium wilting, Septoria, and bacterial blight. The species composition of diseases and the degree of their development were largely influenced by the weather conditions of the growing season. The investigated fungicides Abacus mk.e. (pyraclostrobin, 62.5 + epoxiconazole, 62.5), Amistar Extra 280 SC, (azoxystrobin, 200 + cyproconazole, 80), Acanto Plus 28 c.s. (picoxystrobin, 200 g/l + cyproconazole, 80 g/l), Coronet 300 SC (trifloxystrobin, 100 g/l + tebuconazole, 200 g/l), as well as Impact K preparations, c.s. (flutriafol, 117.5 g/l + carbendazim, 250 g/l) and Koside 2000 w. g. (copper hydroxide, 350 g/kg) at the recommended application rates effectively limited the development of most fungal pathogens. The highest protective effect of 69.8—78.9% of fungicides was shown against downy mildew of soybeans, the lowest — 31.7—42.2% against Alternaria, which had the highest development in comparison with other diseases. Fungicide Koside 2000 w. g. at the level of 67% limited the development of bacterial diseases. The use of fungicides had a positive effect on the yield of soybeans. Due to the limitation of the development of diseases, the soybean yield increased by 21.2—30.3%, depending on the variant of the experiment.
 Conclusions. The use of fungicides significantly affected the limitation of the development of the most common soybean diseases in the Forest-steppe of Ukraine. The effectiveness of The effectiveness of the studied modern fungicides against peronospora, fusarium wilting, septoria was at the level of 60.2—78.9%. Fungicides most effectively controlled the development of downy mildew, less effectively — the development of Alternaria. The limitation of soybean diseases when using fungicides contributed to an increase in its yield by an average of 0.7—1.0 t/ha.

The Plant-Beneficial Rhizobacterium Bacillus velezensis FZB42 Controls the Soybean Pathogen Phytophthora sojae Due to Bacilysin Production.

Soybean root rot caused by the oomycete Phytophthora sojae is a serious soilborne disease threatening soybean production in China. Bacillus velezensis FZB42 is a model strain for Gram-positive plant growth-promoting rhizobacteria and is able to produce multiple antibiotics. In this study, we demonstrated that B. velezensis FZB42 can efficiently antagonize P. sojae. The underlying mechanism for the inhibition was then investigated. The FZB42 mutants deficient in the synthesis of lipopeptides (bacillomycin D and fengycin), known to have antifungal activities, and polyketides (bacillaene, difficidin, and macrolactin), known to have antibacterial activities, were not impaired in their antagonism toward P. sojae; in contrast, mutants deficient in bacilysin biosynthesis completely lost their antagonistic activities toward P. sojae, indicating that bacilysin was responsible for the activity. Isolated pure bacilysin confirmed this inference. Bacilysin was previously shown to be antagonistic mainly toward prokaryotic bacteria rather than eukaryotes. Here, we found that bacilysin could severely damage the hyphal structures of P. sojae and lead to the loss of its intracellular contents. A device was invented allowing interactions between P. sojae and B. velezensis FZB42 on nutrient agar. In this manner, the effect of FZB42 on P. sojae was studied by transcriptomics. FZB42 significantly inhibited the expression of P. sojae genes related to growth, macromolecule biosynthesis, pathogenicity, and ribosomes. Among them, the genes for pectate lyase were the most significantly downregulated. Additionally, we showed that bacilysin effectively prevents soybean sprouts from being infected by P. sojae and could antagonize diverse Phytophthora species, such as Phytophthora palmivora, P. melonis, P. capsici, P. litchi, and, most importantly, P. infestans. IMPORTANCEPhytophthora spp. are widespread eukaryotic phytopathogens and often extremely harmful. Phytophthora can infect many types of plants important to agriculture and forestry and thus cause large economic losses. Perhaps due to inappropriate recognition of Phytophthora as a common pathogen in history, research on the biological control of Phytophthora is limited. This study shows that B. velezensis FZB42 can antagonize various Phytophthora species and prevent the infection of soybean seedlings by P. sojae. The antibiotic produced by FZB42, bacilysin, which was already known to have antibacterial effectiveness, is responsible for the inhibitory action against Phytophthora. We further showed that some Phytophthora genes and pathways may be targeted in future biocontrol studies. Therefore, our data provide a basis for the development of new tools for the prevention and control of root and stem rot in soybean and other plant diseases caused by Phytophthora.