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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Discovery and Fine Mapping of qSCR6.01, a Novel Major QTL Conferring Southern Rust Resistance in Maize.

Southern corn rust (SCR), an airborne disease caused by Puccinia polysora, can severely reduce the yield of maize (Zea mays L.). Using recombinant inbred lines (RILs) derived from a cross between susceptible inbred line Ye478 and resistant Qi319 in combination with their high-density genetic map, we located five quantitative trait loci (QTLs) against SCR, designated as qSCR3.04, qSCR5.07, qSCR6.01, qSCR9.03, and qSCR10.01, on chromosomes 3, 5, 6, 9, and 10, respectively. Each QTL could explain 2.84 to 24.15% of the total phenotypic variation. qSCR6.01, detected on chromosome 6, with the highest effect value, accounting for 17.99, 23.47, and 24.15% of total phenotypic variation in two environments and best linear unbiased prediction, was a stably major resistance QTL. The common confidence interval for qSCR6.01 was 2.95 Mb based on the B73 RefGen_v3 sequence. The chromosome segment substitution lines (CSSLs) constructed with Qi319 as the donor parent and Ye478 as the recurrent parent were used to further verify qSCR6.01 resistance to SCR. The line CL183 harboring introgressed qSCR6.01 showed obvious resistance to SCR that was distinctly different from that of Ye478 (P = 0.0038). Further mapping of qSCR6.01 revealed that the resistance QTL was linked to insertion-deletion markers Y6q77 and Y6q79, with physical locations of 77.6 and 79.6 Mb, respectively, on chromosome 6. Different from previous major genes or QTLs against SCR on chromosome 10, qSCR6.01 was a newly identified major QTL resistance to SCR on chromosome 6 for the first time. Using RIL and CSSL populations in combination, the SCR-resistance QTL research can be dissected effectively, which provided important gene resource and genetic information for breeding resistant varieties.

Characterization and Molecular Mapping of Two Novel Genes Resistant to Pythium Stalk Rot in Maize.

Pythium stalk rot caused by Pythium inflatum is becoming a more and more serious disease in maize, and it has caused severe yield loss in China in recent years. Deployment of resistant maize varieties is the most effective way to control this disease. Searching for the resistant maize germplasm and identifying the resistance genes are the vital processes in the breeding program. The maize inbred line X178 previously showed high resistance to Pythium stalk rot. Thus, this study aimed to reveal the gene(s) resistance to Pythium stalk rot in X178 by resistance inheritance analysis using the derived F2 and F2:3 genetic populations. The results showed that two independently inherited dominant genes, designated RpiX178-1 and RpiX178-2, carried by X178 are responsible for its resistance relative to the susceptible parent Ye107; they are located on regions of maize chromosome (chr.) 1 bin 1.09 and chr. 4 bin 4.08, respectively, and flanked by markers umc2047 and bnlg1671 as well as bnlg1444 and umc1313, respectively, by linkage analysis. Subsequently, RpiX178-1 was finely mapped between markers SSRZ8 and IDP2347, with genetic distances of 0.6 and 1.1 cM, respectively, and the physical distance of the target region was about 700 kb. RpiX178-2 was also further located between markers bnlg1444 and umc2041, with a genetic distance of 2.4 cM. Moreover, we confirmed that the two genes RpiX178-1 and RpiX178-2 were newly identified and different from those genes known on chrs. 1 and 4 according to results of allelism testing. Herein, we newly identified two genes resistant to P. inflatum, which provided important genetic information for resistance to Pythium stalk rot in maize.

Genetic Mapping and Molecular Characterization of a Broad-spectrum Phytophthora sojae Resistance Gene in Chinese Soybean.

Phytophthora root rot (PRR) causes serious annual soybean yield losses worldwide. The most effective method to prevent PRR involves growing cultivars that possess genes conferring resistance to Phytophthora sojae (Rps). In this study, QTL-sequencing combined with genetic mapping was used to identify RpsX in soybean cultivar Xiu94-11 resistance to all P. sojae isolates tested, exhibiting broad-spectrum PRR resistance. Subsequent analysis revealed RpsX was located in the 242-kb genomic region spanning the RpsQ locus. However, a phylogenetic investigation indicated Xiu94-11 carrying RpsX is distantly related to the cultivars containing RpsQ, implying RpsX and RpsQ have different origins. An examination of candidate genes revealed RpsX and RpsQ share common nonsynonymous SNP and a 144-bp insertion in the Glyma.03g027200 sequence encoding a leucine-rich repeat (LRR) region. Glyma.03g027200 was considered to be the likely candidate gene of RpsQ and RpsX. Sequence analyses confirmed that the 144-bp insertion caused by an unequal exchange resulted in two additional LRR-encoding fragments in the candidate gene. A marker developed based on the 144-bp insertion was used to analyze the genetic population and germplasm, and proved to be useful for identifying the RpsX and RpsQ alleles. This study implies that the number of LRR units in the LRR domain may be important for PRR resistance in soybean.

Two Novel er1 Alleles Conferring Powdery Mildew (Erysiphe pisi) Resistance Identified in a Worldwide Collection of Pea (Pisum sativum L.) Germplasms.

Powdery mildew caused by Erysiphe pisi DC. severely affects pea crops worldwide. The use of resistant cultivars containing the er1 gene is the most effective way to control this disease. The objectives of this study were to reveal er1 alleles contained in 55 E. pisi-resistant pea germplasms and to develop the functional markers of novel alleles. Sequences of 10 homologous PsMLO1 cDNA clones from each germplasm accession were used to determine their er1 alleles. The frame shift mutations and various alternative splicing patterns were observed during transcription of the er1 gene. Two novel er1 alleles, er1-8 and er1-9, were discovered in the germplasm accessions G0004839 and G0004400, respectively, and four known er1 alleles were identified in 53 other accessions. One mutation in G0004839 was characterized by a 3-bp (GTG) deletion of the wild-type PsMLO1 cDNA, resulting in a missing valine at position 447 of the PsMLO1 protein sequence. Another mutation in G0004400 was caused by a 1-bp (T) deletion of the wild-type PsMLO1 cDNA sequence, resulting in a serine to leucine change of the PsMLO1 protein sequence. The er1-8 and er1-9 alleles were verified using resistance inheritance analysis and genetic mapping with respectively derived F2 and F2:3 populations. Finally, co-dominant functional markers specific to er1-8 and er1-9 were developed and validated in populations and pea germplasms. These results improve our understanding of E. pisi resistance in pea germplasms worldwide and provide powerful tools for marker-assisted selection in pea breeding.

Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, RpsHC18, in soybean.

KEY MESSAGE: A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS-LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed. Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites-leucine-rich repeat (NBS-LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.

Genetic mapping and development of co-segregating markers of RpsQ, which provides resistance to Phytophthora sojae in soybean.

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

An Emerging Disease Caused by Pseudomonas syringae pv. phaseolicola Threatens Mung Bean Production in China.

An emerging bacterial disease with symptoms resembling those of halo blight is threatening mung bean production in China. This study was conducted to investigate the disease's geographic distribution in China using consecutive multiyear field surveys and to confirm the causative agents' identity. The surveys were conducted in 15 provinces covering seven geographic regions from 2009 to 2014. The survey results revealed that the emerging mung bean disease has rapidly spread and is prevalent in three of the main Chinese geographic regions, which contain more than 90% of the mung-bean-growing areas in China. To confirm the causal agent, diseased mung bean leaves were collected from the surveyed fields and used to isolate the pathogen. A bacterium was consistently isolated from all of the collected leaves. Based on the phenotypic characteristics, the physiological and biochemical properties, pathogenicity tests, and fatty acid composition, in combination with specific polymerase chain reactions and 16S-23S ribosomal DNA sequence analyses, the bacterium was identified as Pseudomonas syringae pv. phaseolicola. To our knowledge, this is the first report of P. syringae pv. phaseolicola causing halo blight on mung bean in China. The results indicate that P. syringae pv. phaseolicola is likely of epidemiological significance on mung bean in China.

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean .

SIZ1 is a small ubiquitin-related modifier (SUMO) E3 ligase that mediates post-translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1-2, including dwarfism, constitutively activated expression of pathogen-related genes, and ABA-sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)-mediated gene silencing decreased heat shock-induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1-2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.