An Emerging Disease of Chickpea, Basal Stem Rot Caused by Diaporthe aspalathi in China.

Chickpea (Cicer arietinum L.) is an important legume crop worldwide. An emerging disease, basal stem rot with obvious wilt symptoms, was observed in the upper part of chickpea plants during the disease survey in Qiubei County of Yunnan Province. Three fungal isolates (ZD36-1, ZD36-2, and ZD36-3) were obtained from the diseased tissue of chickpea plants collected from the field. Those isolates were morphologically found to be similar to Diaporthe aspalathi. Molecular sequence analyses of multiple gene regions (ITS, tef1, tub2, cal, and his3) indicated that the three isolates showed a high identity with D. aspalathi. Pathogenicity and host range tests of the isolates were performed on the original host chickpea and eight other legume crops. The isolates were strongly pathogenic to chickpea and appeared highly pathogenic to soybean, cowpea, and mung bean; moderated or mild pathogenic to adzuki bean and common bean; however, the isolates did not cause symptoms on grass pea (Lathyrus sativus). Diaporthe aspalathi was previously reported as a main pathogen causing the southern stem canker in soybean. To our knowledge, this is the first report of D. aspalathi inducing basal stem rot on chickpea worldwide.

Fine mapping and identification of a Fusarium wilt resistance gene FwS1 in pea.

KEY MESSAGE: A Fusarium wilt resistance gene FwS1 on pea chromosome 6 was identified and mapped to a 91.4 kb region by a comprehensive genomic-based approach, and the gene Psat6g003960 harboring NB-ARC domain was identified as the putative candidate gene. Pea Fusarium wilt, incited by Fusarium oxysporum f. sp. pisi (Fop), has always been a devastating disease that causes severe yield losses and economic damage in pea-growing regions worldwide. The utilization of pea cultivars carrying resistance gene is the most efficient approach for managing this disease. In order to finely map resistance gene, F2 populations were established through the cross between Shijiadacaiwan 1 (resistant) and Y4 (susceptible). The resistance genetic analysis indicated that the Fop resistance in Shijiadacaiwan 1 was governed by a single dominant gene, named FwS1. Based on the bulked segregant analysis sequencing analyses, the gene FwS1 was initially detected on chromosome 6 (i.e., linking group II, chr6LG2), and subsequent linkage mapping with 589 F2 individuals fine-mapped the gene FwS1 into a 91.4 kb region. The further functional annotation and haplotype analysis confirmed that the gene Psat6g003960, characterized by a NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain, was considered as the most promising candidate gene. The encoding amino acids were altered by a "T/C" single-nucleotide polymorphism (SNP) in the first exon of the Psat6g003960, and based on this SNP locus, the molecular marker A016180 was determined to be a diagnostic marker for FwS1 by validating its specificity in both pea accessions and genetic populations with different genetic backgrounds. The FwS1 with diagnostic KASP marker A016180 could facilitate marker-assisted selection in resistance pea breeding in pea. In addition, a comparison of the candidate gene Psat6g003960 in 74SN3B and SJ1 revealed the same sequences. This finding indicated that 74SN3B carried the candidate gene for FwS1, suggesting that FwS1 and Fwf may be closely linked or an identical resistant gene against Fusarium wilt.

Three novel er1 alleles and their functional markers for breeding resistance to powdery mildew (Erysiphe pisi) in pea.

Powdery mildew caused by Erysiphe pisi DC is a global notorious disease on peas. Deploying resistance pea cultivars is the most efficient and environmentally friendly method for the disease control. This study focuses on revealing the resistance genes in three pea germplasms and developing their functional markers for resistance breeding. The identification of resistance genes involved genetic mapping and the sequencing of the PsMLO1 gene. To confirm the hereditary in three reisistant germplasms, they were crossed with susceptible cultivars to generate F1, F2, and F2:3 populations. The F1 generation exhibited susceptibility to E. pisi, while segregation patterns in subsequent generations adhered to the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, indicating that powdery mildew resistance was governed by single recessive gene in each germplasm. Analysis of er1-linked markers and genetic mapping suggested that the resistance genes could be er1 alleles in these germplasms. The multiple clone sequencing results of the three homologous PsMLO1 genes showed they were novel er1 alleles, named er1-15, er1-16, and er1-17, respectively. The er1-15 and er1-16 were caused by 1-bp deletion at position 335 (A) and 429 (T) in exon 3, respectively, while er1-17 was caused a 1-bp insertion at position 248 in exon 3, causing a frame-shift mutation and premature termination of PsMLO1 protein translation. Their respective functional markers KASP-er1-15, KASP-er1-16 and KASP-er1-17 were successfully developed and validated in respective mapping populations and pea germplasms. These results provide valuable tools for pea breeding resistance to E pisi.

Soybean steroids improve crop abiotic stress tolerance and increase yield.

Sterols have long been associated with diverse fields, such as cancer treatment, drug development, and plant growth; however, their underlying mechanisms and functions remain enigmatic. Here, we unveil a critical role played by a GmNF-YC9-mediated CCAAT-box transcription complex in modulating the steroid metabolism pathway within soybeans. Specifically, this complex directly activates squalene monooxygenase (GmSQE1), which is a rate-limiting enzyme in steroid synthesis. Our findings demonstrate that overexpression of either GmNF-YC9 or GmSQE1 significantly enhances soybean stress tolerance, while the inhibition of SQE weakens this tolerance. Field experiments conducted over two seasons further reveal increased yields per plant in both GmNF-YC9 and GmSQE1 overexpressing plants under drought stress conditions. This enhanced stress tolerance is attributed to the reduction of abiotic stress-induced cell oxidative damage. Transcriptome and metabolome analyses shed light on the upregulation of multiple sterol compounds, including fucosterol and soyasaponin II, in GmNF-YC9 and GmSQE1 overexpressing soybean plants under stress conditions. Intriguingly, the application of soybean steroids, including fucosterol and soyasaponin II, significantly improves drought tolerance in soybean, wheat, foxtail millet, and maize. These findings underscore the pivotal role of soybean steroids in countering oxidative stress in plants and offer a new research strategy for enhancing crop stress tolerance and quality from gene regulation to chemical intervention.

Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

Integrated multi-omics analysis reveals insights into Chinese forest musk deer (Moschus berezovskii) genome evolution and musk synthesis.

Among the artiodactyls, male animals belonging to the Family Moschidae have a unique tissue, the musk gland, with the capability of musk synthesis. However, the genetic basis of musk gland formation and musk production are still poorly understood. Here, musk gland tissues from two juvenile and three adult Chinese forest musk deer (Moschus berezovskii) were utilized to analyze genomic evolution events, evaluate mRNA profiles and investigate cell compositions. By performing genome reannotation and comparison with 11 ruminant genomes, three expanded gene families were identified in the Moschus berezovskii genome. Transcriptional analysis further indicated that the musk gland displayed a prostate-like mRNA expression pattern. Single-cell sequencing revealed that the musk gland is composed of seven distinguishable cell types. Among them, sebaceous gland cells and luminal epithelial cells play important roles in musk synthesis, while endothelial cells master the regulation of cell-to-cell communication. In conclusion, our study provides insights into musk gland formation and the musk-synthesizing process.

Integrative transcriptome and proteome analysis reveals maize responses to Fusarium verticillioides infection inside the stalks.

Fusarium stalk rot caused by Fusarium verticillioides is one of the most devastating diseases of maize that causes significant yield losses and poses potential security concerns for foods worldwide. The underlying mechanisms of maize plants regulating defence against the disease remain poorly understood. Here, integrative proteomic and transcriptomic analyses were employed to identify pathogenesis-related protein genes by comparing differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in maize stalks after inoculation with F. verticillioides. Functional enrichment analysis showed that DEGs and DEPs were mainly enriched in glutathione metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. Fourteen DEGs and DEGs that were highly elevated after inoculation with F. verticillioides were confirmed with parallel reaction monitoring and reverse transcription-quantitative PCR, demonstrating the accountability and reliability of proteomic and transcriptomic data. We also assessed the potential roles of defence-related genes ZmCTA1, ZmWIP1, and ZmLOX2, identified from the multi-omics analysis, during the process of F. verticillioides infection through virus-induced gene silencing. The elevation of stalk rot symptomatic characteristics in the silenced plants revealed their contribution to resistance. We further functionally characterized the roles of ZmLOX2 expression in the defence response of maize plants conditioning fungal invasion via the salicylic acid-dependent pathway. Collectively, this study provides a comprehensive analysis of transcriptome and proteome of maize stalks following F. verticillioides inoculation, and defence-related genes that could inform selection of new genes as targets in breeding strategies.

First Report of Botrytis cinerea Causing Stem Rot on Pea (Pisum sativum L.) in China.

Pea (Pisum sativum L.) is one of the most important cool season legumes consumed as vegetable in the world. In March 2022, a severe stem rot was observed on pea cultivars in vegetative stage in Wuhan, Hubei Province, China (30°39' N, 114°66' E). The infection started on the lower stems, and the lesions were water soaked, then girdled the stem, resulting in wilting of the leaves. Eventually, the entire plant died, and some necrotic stems were covered with gray conidia. To investigate the causal agent, small pieces cut from diseased stems were surface sterilized with 2% NaOCl for 1 min, then incubated on potato dextrose agar (PDA) at 25°C for 3 days. Pure cultures were obtained by hyphal tip transfer and five isolates were studied further. Colonies initially appeared white, turned gray from the center, then became taupe with cottony aerial mycelia, and finally black hard, round or irregular sclerotia (0.92 to 5.34 × 0.86 to 4.42 mm, n = 20) developed. The sealing film of several plates were removed after 5 days, and abundant conidia were produced 3 days later. The conidia are terminally arranged at the end of long, grayish branched conidiophores, conidia are unicellular, hyaline and round or elliptical, (9.2 to 11.4 × 6.7 to 9.2 µm, n = 50), and the conidiophores are (10.7 to 13.0 µm × 760 to 1080 µm, n=20) in size. The morphological characteristics were consistent with descriptions of Botrytis cinerea (Li et al., 2016). Genomic DNA of the five isolates was extracted, and the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, heat-shock protein 60 (HSP60) gene, and DNA-dependent RNA polymerase subunit II (RPB2) gene were amplified using the primers described by Aktaruzzaman et al. (2018). The sequences were deposited in GenBank (accession nos. ON533694 and ON566787-ON566790 for ITS; ON600613 to ON600617 for HSP60; ON600608 to ON600612 for G3PDH; ON600603 to ON600607 for RPB2). The BLASTn analysis of these sequences showed that the isolates had high similarity (99 to 100%) with other B. cinerea isolates. A phylogenetic tree was constructed by MEGA11, and our isolates clustered in the B. cinerea clade. In pathogenicity test, 2-week-old seedlings of pea cultivar 'Zhongqin1' were inoculated. Mycelial plugs (5 mm diameter) taken from a 3-day-old colony of each isolate were placed on the axil of a stipule at the 4th node of potted pea plants (n=5 per isolate), and PDA plugs were placed on the same location of control (n=3). Inoculated and control plants were kept in a humid plastic box at 23°C for 2 days, and then placed in a glasshouse. Symptoms with water-soaked lesions were observed on the inoculated plants after 2 days, stems showed soft rot and broke off after 3 to 5 days, disease symptoms similar to those in the field, while the controls remained healthy. The pathogen was re-isolated from the affected stems, fulfilling Koch's postulates. B. cinerea had been reported to cause foliar, pod, seed and stem rot of pea after flowering in many pea production regions in the world (Kraft and Pfleger, 2001). Pea was recorded as a host of B. cinerea in Zhejiang, Sichuan and Yunnan Provinces (Tai, F. L. 1979; Zhuang, W.-Y. 2005; Zhang, Z. 2006.), but there has been no detailed disease description and identification of pathogen. To our knowledge, this is the first report of B. cinerea causing stem rot on pea in vegetative stage in China. Since B. cinerea can infect pea at any developmental stage, it could have a high economic impact as green pea production increases in China.

Berkeleyomyces rouxiae is a causal agent of root rot complex on faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is an important food and feed legume crop in the world. The root rot complex caused by various pathogens is a main constraint in faba bean production. In April 2021, a severe disease of faba bean with symptoms of black necrosis on roots occurred in experimental fields at the Linxia Institute of Agricultural Sciences, Gansu Province, China. This study aimed to identify the pathogen and evaluate the resistance of faba bean cultivars. The pathogen was isolated from infected soils, and five representative isolates were identified as Berkeleyomyces rouxiae based on morphological characteristics, pathogenicity, and molecular phylogenetic analyses. A host range test showed that chickpea, common bean, cowpea, mung bean, rice bean, lentil, and hyacinth bean were susceptible hosts of the faba bean isolate, whereas adzuki bean, pea, and soybean were non-susceptible hosts, and maize and wheat were non-hosts. Identification of resistance among 36 faba bean cultivars was carried out, and six cultivars were found to be moderately resistant to B. rouxiae. In this study, we first reported black root rot on faba bean caused by B. rouxiae, confirmed and expanded the host range of B. rouxiae, and identified resistant faba bean cultivars.

Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces.

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.

Identification of a Fusarium ear rot resistance gene in maize by QTL mapping and RNA sequencing.

Fusarium ear rot (FER) caused by Fusarium verticillioides is a prevalent maize disease. To comprehensively characterize the genetic basis of the natural variation in FER resistance, a recombinant inbred line (RIL) population was used to map quantitative trait loci (QTL) for FER resistance. A total of 17 QTL were identified by linkage mapping in eight environments. These QTL were located on six chromosomes and explained 3.88-15.62% of the total phenotypic variation. Moreover, qFER1.03 had the strongest effect and accounted for 4.98-15.62% of the phenotypic variation according to analyses of multiple environments involving best linear unbiased predictions. The chromosome segment substitution lines (CSSLs) derived from a cross between Qi319 (donor parent) and Ye478 (recurrent parent) were used to verify the contribution of qFER1.03 to FER resistance. The line CL171, which harbored an introgressed qFER1.03, was significantly resistant to FER. Further fine mapping of qFER1.03 revealed that the resistance QTL was linked to insertion/deletion markers InDel 8 and InDel 2, with physical distances of 43.55 Mb and 43.76 Mb, respectively. Additionally, qFER1.03 differed from the previous resistance QTL on chromosome 1. There were three annotated genes in this region. On the basis of the RNA-seq data, which revealed the genes differentially expressed between the FER-resistant Qi319 and susceptible Ye478, GRMZM2G017792 (MPK3) was preliminarily identified as a candidate gene in the qFER1.03 region. The Pr-CMV-VIGS system was used to decrease the GRMZM2G017792 expression level in CL171 by 34-57%, which led to a significant decrease in FER resistance. Using RIL and CSSL populations combined with RNA-seq and Pr-CMV-VIGS, the candidate gene can be dissected effectively, which provided important gene resource for breeding FER-resistant varieties.

Disease Resistance and Molecular Variations in Irradiation Induced Mutants of Two Pea Cultivars.

Induced mutation is useful for improving the disease resistance of various crops. Fusarium wilt and powdery mildew are two important diseases which severely influence pea production worldwide. In this study, we first evaluated Fusarium wilt and powdery mildew resistance of mutants derived from two elite vegetable pea cultivars, Shijiadacaiwan 1 (SJ1) and Chengwan 8 (CW8), respectively. Nine SJ1 and five CW8 M3 mutants showed resistant variations in Fusarium wilt, and the same five CW8 mutants in powdery mildew. These resistant variations were confirmed in M4 and M5 mutants as well. Then, we investigated the genetic variations and relationships of mutant lines using simple sequence repeat (SSR) markers. Among the nine effective SSR markers, the genetic diversity index and polymorphism information content (PIC) values were averaged at 0.55 and 0.46, which revealed considerable genetic variations in the mutants. The phylogenetic tree and population structure analyses divided the M3 mutants into two major groups at 0.62 genetic similarity (K = 2), which clearly separated the mutants of the two cultivars and indicated that a great genetic difference existed between the two mutant populations. Further, the two genetic groups were divided into five subgroups at 0.86 genetic similarity (K = 5) and each subgroup associated with resistant phenotypes of the mutants. Finally, the homologous PsMLO1 cDNA of five CW8 mutants that gained resistance to powdery mildew was amplified and cloned. A 129 bp fragment deletion was found in the PsMLO1 gene, which was in accord with er1-2. The findings provide important information on disease resistant and molecular variations of pea mutants, which is useful for pea production, new cultivar breeding, and the identification of resistance genes.

Identification of Causal Agent Inciting Powdery Mildew on Common Bean and Screening of Resistance Cultivars.

Powdery mildew is one of the severe diseases on common bean in Southwestern China, but the identity of the pathogen inciting this disease is unclear. The objective of this study was to identify the causal agent of common bean powdery mildew and to screen resistant cultivars. The pathogen was identified through morphological identification, molecular phylogenetic analysis, and pathogenicity tests. Resistance of common bean cultivars was evaluated by artificial inoculation at the seedling stage. The common bean powdery mildew isolate CBPM1 was obtained after pathogen isolation and purification. Morphological identification confirmed that the isolate CBPM1 belonged to the Oidium subgenus Pseudoidium and germinated Pseudoidium-type germ tubes. Molecular phylogenetic analysis showed that the isolate CBPM1 and Erysiphe vignae isolates from different hosts were clustered into a distinct group. The pathogenicity and host range tests revealed that the isolate CBPM1 was strongly pathogenic to common bean, multiflora bean, lablab bean, cowpea, and mung bean, but not to soybean, adzuki bean, pea, faba bean, chickpea, lentil, pumpkin, and cucumber. In addition, 54 common bean cultivars were identified for resistance to powdery mildew, and 15 were resistant or segregant. Based on the morphological, molecular and pathogenic characteristics, the causal agent of common bean powdery mildew was identified as E. vignae. This is the first time E. vignae has been confirmed on common bean. Cultivars with different resistance levels were screened, and these cultivars could be used for disease control or the breeding of new resistant cultivars.

Production of new wheat-A. cristatum translocation lines with modified chromosome 2P coding for powdery mildew and leaf rust resistance.

Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a relative of wheat, carries desirable genes associated with high yield, disease resistance, and stress resistance, which is an important resource for wheat genetic improvement. The long arm of A. cristatum chromosome 2P carries favorable genes conferring powdery mildew and leaf rust resistance, and two wheat-A. cristatum 2P translocation lines, 2PT3 and 2PT5, with a large segment of 2P chromatin were obtained. In this study, 2PT3 and 2PT5 translocation lines with powdery mildew and leaf rust resistance genes were used to induce translocations of different chromosomal sizes via ionizing radiation. According to cytological characterization, 10 of those plants were new wheat-A. cristatum 2P small-chromosome segment translocation lines with reduced 2P chromatin, and 6 plants represented introgression lines without visible 2P chromosomal fragments. Moreover, four lines were resistant to both powdery mildew and leaf rust, while two lines were resistant only to leaf rust.

Transcriptome Analysis of Resistance to Fusarium Wilt in Mung Bean (Vigna radiata L.).

Fusarium wilt is a destructive soil-borne disease that threatens the production of mung bean. Mung bean lines Zheng8-4 and Zheng8-20 show high resistance and high susceptibility to Fusarium wilt, respectively. Transcriptome analysis was carried out to identify candidate genes involved in Fusarium wilt resistance using Zheng8-4 and Zheng8-20 at 0, 0.5, 1, 2, and 4 days post inoculation (dpi). Differential expression analysis showed that 3,254 genes responded to pathogen infection and were differentially expressed in the resistant and susceptible lines. Weighted gene co-expression network analysis (WGCNA) was also performed to identify five modules highly correlated with Fusarium wilt resistance, in which 453 differentially expressed genes (DEGs) were considered likely to be involved in Fusarium wilt resistance. Among these DEGs, we found 24 genes encoding resistance (R) proteins, 22 encoding protein kinases, 20 belonging to transcription factor families, 34 encoding proteins with oxidoreductase activity, 17 involved in stimulation/stress responses, and 54 annotated to pathogen resistance-related pathways. Finally, 27 annotated genes were further selected as candidate genes of Fusarium wilt resistance in mung bean. This study identifies novel potential resistance-related genes against Fusarium wilt and provides a theoretical basis for further investigation of Fusarium wilt resistance in mung bean breeding.

Genome Sequence Data of Three Formae Speciales of Phytophthora vignae Causing Phytophthora Stem Rot on Different Vigna Species.

Phytophthora vignae is an important oomycete pathogen causing Phytophthora stem rot on some Vigna spp. Three P. vignae isolates obtained from mung bean, adzuki bean, and cowpea exhibited high similarities in morphology and physiology but are specialized to infect different hosts. Here, we report the first de novo assembly of the draft genomes of three P. vignae isolates, which were performed using the PacBio SMRT Sequel platform. This study will extend the genomic resource available for the Phytophthora genus and provide a good foundation for further research on comparative genomics of Phytophthora spp. and interaction mechanism between hosts and pathogens.

First Report of Maize Stalk Rot Caused by Fusarium kyushuense in China.

During 2017 to 2019, a field survey for maize stalk rot was conducted in 21 counties (districts) across the Guangxi province of China. This disease caused yield losses ranging from 20% to 30%. Maize plants with stalk rot were collected during the late milk stage and pieces of diseased pith tissue were cultured as previously described (Shan et al. 2017). Fungal colonies and mycelia with morphological characteristics of Fusarium species were subcultured onto fresh potato dextrose agar (PDA) and carnation leaf agar (CLA) plates. Based on morphological characteristics and molecular detection by amplification of Fusarium genus-specific primers (Duan et al. 2016), 39 Fusarium isolates were identified. Among them, five isolates from Du'an, Pingguo, Debao, and Daxin had abundant, pale orange to yellow aerial mycelium with deep red pigments when grown on PDA (Fig. 1A; 1B). The average growth rate was 8.0 to 12.0 mm per day at 25°C in the dark. The fungi produced two types of spores on CLA. Microconidia were ovoid to clavate, generally 0- to 3-septate, and 4.6 to 9.4 µm in length (n = 30) (Fig. 1D); Macroconidia were slightly curved with an acute apical cell, mostly 3- to 4- septate, and 19.4 to 38.2 µm in length (n = 30) (Fig. 1C). No chlamydospores were observed. These five isolates were initially identified as Fusarium kyushuense based on morphological features. PCR was performed to amplify three phylogenetic genes (TEF1-α, RPB1, and RPB2) (O'Donnell et al. 1998) and species specific primers kyuR1F/kyuR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3/5-TCCAATGGACTGGGCAGCCAAAACACC-3), kyuR2F/kyuR2R (5-CAGATATACATTTGCCTCGACAC-3/5-TACTTGAGCACGGAGCTTG-3) were used to confirm species identity. The obtained sequences were deposited in GenBank under the accession numbers MT997084, MT997080, MT997081 (TEF1-α); MT550012, MT997085, MT997086 (RPB1); MT550009, MT997089, and MT997090 (RPB2), respectively. Using BLAST, sequences of TEF1-α, RPB1, and RPB2 of the isolates were 99.33% (MH582297.1) to 100% (MG282364.1) similar to those of F. kyushuense strains (Supplementary Table 1). Based on phylogenetic analysis with maximum likelihood methods using tools of the website of CIPRES (http://www.phylo.org), isolates GX27, GX167, and GX204 clustered with F. kyushuense with 100% bootstrap support (Fig. 2). The pathogenicity of the three isolates was tested using young seedlings and adult plants as previously described with modification (Ye et al. 2013; Zhang et al. 2016). The primary roots of three-leaf-old seedlings were inoculated by immersing the roots into a 1 × 106 macroconidia solution, incubating for 6 h at 25°C, and transferring to normal growth conditions (26°C, 16 h light/22°C, 8 h dark). The second or third internode above the soil surface of flowering stage plants grown in a greenhouse was bored with a Bosch electric drill to make a hole (ca. 8 mm in diameter) and inoculated with 0.5 mL of mycelia plug then sealed with petrolatum. The inoculum was created by homogenizing five plates of flourish hyphal mats (approximately 125 mL) with kitchen blender and adjusting to a final volume of 200 mL with sterilized ddH2O. No symptoms were observed in the seedlings or adult plants that were mock-inoculated with PDA plugs. Three days post-inoculation (dpi), roots of the infected seedling turned dark-brown and shrunk and the leaves wilted (Fig. 1E). Typical stalk rot symptoms observed in the inoculated plants were premature wilting of entire plant and hollow and weak stalks, leading to lodging; the longitudinal section of the internodes exhibited obvious dark brown necrosis and reddish discoloration at 14 dpi and 30 dpi, respectively (Fig. 1F). Fusarium kyushuense was re-isolated from the inoculated stalk lesions but not from the control. This is the first record of stalk rot caused by F. kyushuense on maize plants in China. However, F. kyushuense is known to cause maize ear rot in China (Wang et al. 2014) and can produce type A and type B trichothecene mycotoxins in kernels (Aoki and O'Donnell 1998). The occurrence of maize stalk rot and ear rot caused by F. kyushuense should be monitored in China due to the potential risk for crop loss and mycotoxin contamination.

A Novel Disease of Mung Bean, Phytophthora Stem Rot Caused by a New Forma Specialis of Phytophthora vignae.

An emerging soilborne disease resembling Phytophthora stem rot was observed on mung bean plants grown in Anhui, China. To identify the causal agent, diseased plants and soil samples from 13 fields were collected to isolate the pathogen. Twenty-two Phytophthora isolates were recovered from the samples and detailed identification was conducted. Based on morphological and molecular characterizations, all of the isolates were consistently identified as P. vignae. Phylogenetic analysis using eight nuclear loci sequences of the internal transcribed spacer region, rRNA gene large subunit, a partial sequence of the ß-tubulin gene, translation elongation factor 1α, 60S ribosomal protein L10, the enolase gene, heat shock protein 90, and triose phosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase and a mitochondrial locus cytochrome c oxidase subunit I revealed that the mung bean isolates grouped in the same clade as P. vignae and its two formae speciales, P. vignae f. sp. adzukicola and P. vignae f. sp. vignae. A host specificity test showed that the mung bean isolates of P. vignae were pathogenic toward mung bean with a much stronger virulence and toward adzuki bean with a relatively weak virulence, but they were nonpathogenic to the other tested legume crops, including soybean, cowpea, pea, common bean, faba bean, and chickpea. The host range of mung bean isolates significantly differs from those of P. vignae f. sp. adzukicola and P. vignae f. sp. vignae based on our results and on previous studies. Thus, the pathogen causing Phytophthora stem rot of mung bean is proposed as a new forma specialis of P. vignae, designated as P. vignae f. sp. mungcola.

First Report of Paramyrothecium foliicola Causing Leaf Spot on Vigna radiata L. in China.

Mung bean (Vigna radiata L.) is an important legume crop cultivated widely in China (Nair et al. 2013). In September 2018, a severe foliar disease occurred on some mung bean cultivars (Jilv0816, Baolv200810-1, Liaolv10L708-5, and Zhonglv5) in Shijiazhuang (38°03'N, 114°29'E), Hebei Province, China. Initially, lesions were circular to irregular, with dark brown margins and pale centers (Supplementary Fig.1). Later, tiny dark stroma with oval or irregular shape were observed on spots. The infected field was about 0.067 hectare with 50-70% disease incidence, but with no significant yield losses. Several leaves with necrotic spots were collected and cut into 2-3-mm pieces, surface sterilized with 2% NaClO for 2 min, rinsed three times in sterile distilled water, and incubated on potato dextrose agar (PDA) at 25ºC in darkness for 7 days. Three of 10 obtained single spore isolates, QB1, QB2 and QB3, were used for further studies. Colonies had abundant white aerial mycelia and produced black sporodochia bearing masses of viscid spores on PDA after 7-10 days. Conidia were aseptate, hyaline, and cylindrical, with the size of 5.6-7.5 µm × 1.6-3.3 µm (n=50). Conidiophores branched repeatedly. These morphological characteristics resembled that of Paramyrothecium-like isolates (Lombard et al. 2016). Given that P. roridum, P. foliicola, and P. nigrum were all reported to cause leaf spot on leafy vegetables and ornamental crops, five loci (the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), ß-tubulin (tub2), 28S rRNA (LSU) and calmodulin (cmdA)) were amplified and sequenced for molecular analysis (Mati et al. 2019). The resulting sequences were deposited in GenBank under accession numbers: MK335967, MT415351-MT415364. Among the five loci, ITS and LSU sequences showed 99-100% (584/590, 545/546 base pairs) similarity with P. foliicola type strain CBS113121 (NR_145074.1; KU846324.1) by BLASTn analysis, while the tef1, tub2, and cmdA sequences exhibited high identity (99%) (398/404 bp, 323-324/326 bp, 555-558/560 bp) with P. foliicola strain Bas4_m2 (MH939239.1; MH824739.1; MH807772.1) (Mati et al. 2019). Phylogenetic tree of the five concatenated loci showed that our isolates cluster with P. foliicola, although they show slight difference from other P. foliicola strains (Supplementary Fig.2). Based on morphology and molecular analysis, the pathogen was identified as P. foliicola. Pathogenicity tests of the three isolates were performed by spraying 2 ml of 1.0 × 106/ml spore suspension on each three-week-old seedlings of mung bean cultivar 'Jilv 7' (n=5 for each isolate), whereas the controls were inoculated with sterile water (n=3). All inoculations were incubated in a moist chamber at 25ºC with a 12h light cycle. The experiment was repeated twice. After 7 to 10 days, symptoms with necrotic brown spots were observed on plants inoculated with P. foliicola, but not on controls. The pathogen was reisolated from randomly selected diseased leaves and identified as P. foliicola by morphology and DNA sequencing of tub2 and cmdA loci. No pathogens were isolated from controls. Although P. roridum has been reported to cause mung bean leaf spot in India (Singh and Shukla 1997; Singh and Narain 2008), to our knowledge, this is the first report of P. foliicola causing leaf spot on mung bean in China. This finding suggests a potential threat to mung bean production in China and further studies should focus on epidemiology and control of this disease.

Fine Mapping, Candidate Gene Identification and Co-segregating Marker Development for the Phytophthora Root Rot Resistance Gene RpsYD25.

Phytophthora root rot (PRR) caused by Phytophthora sojae is a serious disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Rps genes. Soybean cultivar Yudou25 can effectively resist pathotypes of P. sojae in China. Previous studies have mapped the Rps gene in Yudou25, RpsYD25, on chromosome 3. In this study, at first RpsYD25 was located between SSR markers Satt1k3 (2.2 cM) and BARCSOYSSR_03_0253 (4.5 cM) by using an F2:3 population containing 165 families derived from Zaoshu18 and Yudou25. Then the recombination sites were identified in 1127 F3:4 families derived from Zaoshu18 and Yudou25 using the developed PCR-based SNP, InDel and SSR markers, and RpsYD25 was finely mapped in the a 101.3 kb genomic region. In this region, a zinc ion binding and nucleic acid binding gene Glyma.03g034700 and two NBS-LRR genes Glyma.03g034800 and Glyma.03g034900 were predicted as candidate genes of RpsYD25, and five co-segregated SSR markers with RpsYD25 were identified and validated to be diagnostic markers. Combined with the resistance reaction to multiple P. sojae isolates, seven of 178 soybean genotypes were detected to contain RpsYD25 using the five co-segregated SSR markers. The soybean genotypes carrying RpsYD25 and the developed co-segregated markers can be effectively applied in the breeding for P. sojae resistance in China.