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

    Affiliations
    Authors and Affiliations
    • Feifei Sun1 2
    • Suli Sun1
    • Yong Yang3
    • Bin Zhou3
    • Canxing Duan1
    • Weixing Shan2
    • Zhendong Zhu1
    1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China
    2. 2College of Agronomy, Northwest Agriculture and Forestry University, Yangling 712100, P. R. China
    3. 3Crop Institute of Anhui Academy of Agricultural Sciences, Hefei 230031, P. R. China

    Published Online:https://doi.org/10.1094/PDIS-07-20-1513-RE

    Abstract

    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 β-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.

    Mung bean (Vigna radiata L.) is an important grain legume crop. It originates from the Indian subcontinent and is now cultivated throughout southern and eastern Asia, Central Africa, South and North America, and Australia (Dahiya et al. 2015; Nair et al. 2013; Schafleitner et al. 2015). Because mung bean is a short-duration crop and can improve soil fertility by fixing atmospheric nitrogen, it is often incorporated into various multicrop and intercropping systems (Singh et al. 2014). Mung bean is widely grown in the Northeast (Inner Mongolia, Jilin), Middle (Hebei, Anhui), and Southwest (Chongqing, Sichuan) of China, which makes it the world’s second largest mung bean producer next to India (Li et al. 2016, 2017; Nair et al. 2012; Wang et al. 2009). Biotic stresses, including viral, bacterial, and fungal diseases, insect pests, and weeds, are major factors limiting the yield and quality of mung bean (Nair et al. 2012). In China, >20 diseases, including several newly discovered diseases such as mung bean charcoal rot, gray mold, Fusarium wilt, and Verticillium wilt, have been documented on mung bean (Cui et al. 2014; Li et al. 2016; Sun et al. 2016, 2017; Yin et al. 2016; Yue et al. 2015; Zhang et al. 2011; Zhu and Duan 2012).

    The genus Phytophthora contains many species that are destructive pathogens of many plants. Several Phytophthora species can cause seed rot, pre- and postemergence damping-off, and stem and root rot in various legume crops (Chang et al. 2017). Examples include P. sojae, which causes root and stem rot in soybean (Schmitthenner 1985; Tyler 2007); P. vignae, the causal agent of stem and root rot of cowpea and adzuki bean (Kitazawa et al. 1978, 1979; Tsuchiya and Kodama 1978; Tsuchiya et al. 1986); P. pisi, responsible for root rot of pea and faba bean in southern Sweden (Heyman et al. 2013); and P. phaseoli, which causes downy mildew of lima bean (Thaxter 1889).

    Molecular techniques have greatly enhanced the identification and circumscription of Phytophthora species. Based on multilocus phylogenies containing nuclear and mitochondrial genetic markers, species in the genus Phytophthora were divided into 12 phylogenetic clades (Blair et al. 2008; Jung et al. 2017; Kroon et al. 2004; Martin et al. 2014; Yang et al. 2017). Of these clades, some were further divided into several different subclades, such as clade 7 in which four subclades (7a, 7b, 7c and 7d) were included (Yang et al. 2017). P. vignae was clustered in subclade 7b, together with P. sojae, P. pisi, and P. melonis.

    Phytophthora species differ in host range. Some are destructive to hundreds of plant species, whereas others only infect several plants (Erwin and Ribeiro 1996; Liu et al. 2016). Of these Phytophthora species, some can be further divided into formae speciales. For example, P. vignae has been identified for two formae speciales according to host specificity tests: P. vignae f. sp. vignae and P. vignae f. sp. adzukicola (Tsuchiya et al. 1986). These two formae speciales have a similar morphology but distinctly different pathogenicity and virulence toward different hosts. The P. vignae f. sp. vignae isolates were virulent to cowpea but showed no pathogenicity to adzuki bean and mung bean (Dilantha Fernando and Linderman 1993; Tsuchiya et al. 1986), or exhibited only weak virulence to individual adzuki bean cultivars (Kitazawa et al. 1979), whereas P. vignae f. sp. adzukicola isolates could not only infect adzuki bean but also show pathogenicity to some cowpea and mung bean cultivars (Kitazawa et al. 1979; Zhu and Wang 2003).

    In China, Phytophthora diseases on legumes were reported on cowpea in Guangzhou, Zhejiang, Jiangsu, and Anhui provinces and Shanghai City during the 1980s and 1990s, and the pathogens were identified as P. vignae (Cheng and Wei 1999; Huang and Qi 1984; Yang et al. 1989). In 2003, Phytophthora stem rot was discovered on adzuki bean in Jiamusi City, Heilongjiang Province, China, and the pathogen was confirmed to be P. vignae f. sp. adzukicola (Zhu and Wang 2003). However, there have been no reports of Phytophthora species causing diseases on mung bean to date.

    During field surveys for mung bean diseases, a symptom resembling Phytophthora stem rot was observed in Mingguang City, Anhui Province, China, in August 2015. This disease was also observed in some mung bean fields in the following years. The present study was therefore performed to identify the causal agent by (i) morphological and molecular characterizations, (ii) pathogenicity and host specificity tests, and (iii) phylogenetic analysis using multilocus sequences, including eight nuclear loci sequences of the internal transcribed spacer (ITS) region, rRNA gene large subunit (LSU), a partial sequence of the β-tubulin (β-tubulin) gene, translation elongation factor 1α (EF1-α), 60S ribosomal protein L10 (60SL10), the enolase (Enl) gene, heat shock protein 90 (HSP90), and triose phosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase (TigA) and a mitochondrial locus cytochrome c oxidase subunit I (cox1).

    Materials and Methods

    Field survey and diseased sample collection.

    Field surveys were conducted in 13 mung bean fields in Mingguang City (northern latitude of 32°80′, eastern longitude of 118°26′), Anhui Province, China. Diseased plants with Phytophthora stem rot symptoms and soil samples from the rhizosphere of infected plants were collected to isolate the pathogen. The field soil samples were collected by pooling 5 to 10 subsamples from each field into one composite sample per field, and they were dried at an average room temperature of 26°C (23 to 30°C), ground into powder using a pestle, and then filtered with a sieve (60-mesh, 0.3 mm; GB6003-97) before they were used for the leaf disc-baiting assay.

    Pathogen isolation.

    Isolations were made from infected tissues of diseased plants by washing the tissues under tap water and cutting them into several 2- to 3-mm-thick segments. After surface disinfection with 2% NaClO solution for 2 min, the tissues were rinsed three times in sterile distilled water, dried on sterilized filter paper, and placed on the Phytophthora-selective media PBNIC containing 0.054 g of pentachloronitrobenzene, 0.01 g of benlate, 0.1 g of neomyclin sulfate, 0.04 g of iprodione, 0.01 g of chloramphenicol, and 0.02 g of hymexazole (added after autoclaving) in 1,000 ml of V8 agar (V8A; 20% V8 juice, 2% agar, and 0.03% CaCO3) (Dorrance et al. 2016; Wang et al. 1998). The plates were incubated at 25°C in the dark for 3 or 4 days. Isolations from field soil samples were conducted using a leaf disc-baiting technique previously applied to other Phytophthora species (Scanu et al. 2014; Zhu et al. 2003). Thirty grams of each composite field soil sample was dispensed into a Petri dish and moistened with 15 ml of sterile water. The plates were incubated in the dark upside down at 24°C for 7 days. Subsequently, distilled water was added until it was 6 mm higher than the soil level, and leaf discs from the mung bean cultivar Jilv 7 were placed onto water to bait Phytophthora species. After 6 h, the baited leaf discs were transferred to another Petri dish containing distilled water and were incubated in the dark for 36 to 48 h at room temperature. The leaf discs were subsequently inoculated onto the stem of mung bean cultivar Jilv 7, which was wounded using a syringe needle, and the inoculated plants were incubated for 2 or 3 days at 25°C in a moist cabinet. Isolations were made from inoculated symptomatic plants, as described above for isolations from plant tissues. Isolates were tentatively identified as Phytophthora species based on colony morphology such as uniform edges and sparse aerial mycelium on the selective medium. Purification of the isolates was performed through hyphal tip culturing. All isolates were grown on V8A for species identification based on morphological criteria (i.e., antheridia were amphigynous or paragynous; oospores were aplerotic or nearly plerotic; sporangia were papillate, semipapillate, or nonpapillate; shapes and sizes of oogonia, oospores, and sporangia; and presence or absence of chlamydospores and hyphal swellings) (Kroon et al. 2012). Ten isolates (PV1, PV2 from diseased plants, and PVM6, PVM7, PVM8, PVM10, PVM13, PVM15, PVM18, and PVM24 from soil samples) were randomly selected for morphological characterization.

    Morphological characterization observation.

    The 10 isolates were incubated at 25°C in the dark for 2 weeks on V8A plates and any sexual structures, such as oospores, oogonia, and antheridia, were observed and measured under a microscope (Olympus 31X) with the UV-C confocal imaging system (UVTEC).

    Sterile pond water was used to induce the formation of asexual sporangia (Falloon 1982). Agar plugs (5 mm in diameter) were taken from the edge of 3- to 4-day-old colonies grown on V8A and placed in a 60-mm-diameter Petri dish flooded with pond water for 2 days at 25°C. Sporangia and zoospores were observed and measured under a microscope. Thirty representatives of each sexual or asexual structure were randomly selected from each isolate to calculate the mean and range of size.

    Pathogenicity test.

    A pathogenicity test of all obtained isolates was performed on their original host, mung bean, using the hypocotyl wound inoculation method previously applied to P. sojae (Haas and Buzzell 1976; Zhu et al. 2000). Inoculum was prepared by incubating the isolates on 20% V8A medium at 25°C for 7 days until the mycelium nearly covered the entire Petri dish. A mycelial slurry was generated by mixing the colonized agar together. This slurry was pushed through a 20-ml syringe to inoculate the mung bean plants.

    Ten seeds of mung bean cultivar Jilv 7 were planted per paper cup (500 ml) filled with vermiculite. The planted cups were maintained in a greenhouse at 25°C under a 12-h photoperiod and watered regularly every 3 to 4 days until the primary leaves of the seedlings were fully unfolded. After wounding the hypocotyls of seedlings approximately 1 cm below the cotyledon with a sterilized syringe needle, 0.5 ml of the above-mentioned mycelial slurry was injected into the wound. Controls were inoculated using fresh V8A according to the same procedure. All inoculated plants were arranged in a completely randomized design in a cabinet containing a humidifier for 48 h at 28°C, after which the plants were maintained in a greenhouse at 25 to 28°C for another 2 or 3 days. The number of dead plants was scored 5 days postinoculation. Three replications were conducted for each isolate and the experiment was repeated twice.

    Host range test.

    A host range test was conducted on mung bean and seven other legumes, including soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum), chickpea (Cicer arietinum), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), and faba bean (Vicia faba), using all of the obtained mung bean isolates and control isolates P. vignae f. sp. adzukicola (PaV1) and P. vignae f. sp. vignae (PcV2), which were obtained from our laboratory and the Plant Protection Institute of the Fujian Academy of Agricultural Sciences, respectively. Ten cultivars were used for each of the mung bean, adzuki bean, and cowpea hosts (Table 1), and three cultivars were used each of the remaining crop species. The inoculation method and incubation conditions used were the same as described above for the pathogenicity test. In each experiment, 10 plants of each host cultivar were subjected to each treatment in a completely randomized design. Results were recorded 5 days postinoculation and expressed as the percent death rate. The latter was calculated by the following formula: Death rate = Dead plants/Total plants inoculated in each pot × 100%. All experiments were repeated twice.

    Table 1. Response of three legume crops to 24 Phytophthora vignae isolates (22 isolates from mung bean and two P. vignae formae speciales, PaV1 and PcV2) after inoculation under greenhouse conditions

    Statistical analysis.

    The host range trial was analyzed using the general linear model in SAS Analyst software (version 9.2; SAS Institute Inc.). Data on the percentage of seedling death rate were transformed to fit a normal distribution. The transformed death rate data were subjected to a two-way analysis of variance (ANOVA) to investigate the isolate and cultivar effects as well as their interaction. The means of significant virulence difference between isolates were compared using Student’s t least significant difference at a 5% significance level.

    DNA extraction and PCR amplification.

    Mycelia of all mung bean isolates and two other P. vignae formae speciales (PaV1 and PcV2) were grown for 5 days on V8A medium at 25°C, collected with a sterilized scalpel, and ground into a fine powder in liquid nitrogen. Genomic DNA was extracted from the powdered mycelia using the cetrimonium bromide method (Doyle 1987). DNA concentrations were determined with a UV-visible spectrophotometer (Q5000) and genomic DNA was stored at −20°C until further use.

    PCR was conducted using primer pairs ITS4/ITS5 for the rRNA ITS (White et al. 1990), NL1/NL4 for rRNA LSU (O’Donnell 1993), ELONGF1_for/ELONGR1_rev for EF1-α (Kroon et al. 2004), TUBUR2_for/TUBUR1_rev for β-tubulin (Kroon et al. 2004), 60SL10_for/60SL10_rev for 60SL10 (Blair et al. 2008), Enl_for/Enl_rev for Enl (Blair et al. 2008), HSP90_F1/HSP90_R1 for HSP90 (Blair et al. 2008), Tig_for/Tig_rev for TigA (Blair et al. 2008), and OomCoxI-Levlo/OomCoxI-Levup for cox1 (Robideau et al. 2011). The latter gene region was the only mitochondrial locus, and the others were all nuclear loci.

    PCR reactions were carried out in a Gene Amp 9700 thermal cycler (Applied Biosystems) in 25-µl volumes consisting of 12.5 µl of PCR Master Mix (Tiangen), 2 µl (10 µM) each of forward and reverse primers, 1 µl of genomic DNA (50 ng), and 7.5 µl of sterile water. The cycling protocol was as follows: an initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at the respective optimal temperature for each genetic marker for 30 s, and extension at 72°C for 60 s, with a final extension of 72°C for 10 min. The annealing temperatures for each of the gene regions were 55, 55, 60, 60, 53, 60, 62, 64, and 52°C for ITS, LSU, EF1-α, β-tubulin, 60SL10, Enl, HSP90, TigA, and cox1, respectively. All PCR products were analyzed by gel electrophoresis on a 1% agarose gel with 1× Tris/borate/EDTA buffer, and purified products were sequenced in both directions by Sangon Biotech. The generated sequences were compared using the BLAST program against data in the NCBI database. Only sequences from peer-reviewed published articles were considered for reliable species identification.

    Phylogenetic analysis.

    Sequences of the ITS, LSU, cox1, β-tubulin, EF1-α, 60SL10, Enl, HSP90, and TigA regions of all recovered mung bean isolates and two P. vignae formae speciales reference isolates, along with other sequences of Phytophthora species in clade 7 and two outgroup sequences from P. phaseoli and P. infestans in clade 1, were aligned using ClustalW software following the default settings displayed in MEGA5 (Tamura et al. 2011). The nine loci sequences of P. vignae isolate P3019 were downloaded from GenBank and used as type strain sequences. Isolate P3019 was also used in other studies as the type strain (Blair et al. 2008; Martin et al. 2014). Phylograms were constructed using the concatenated nuclear sequence dataset (ITS-LSU-β-tubulin-EF1-α-60SL10-Enl-HSP90-TigA). Alignments were manually edited to trim unaligned regions at both ends and remove all ambiguous positions for each sequence pair in MEGA5 (Ortu et al. 2013). Evolutionary analyses were also conducted in MEGA5 using maximum parsimony and maximum likelihood methods with 1,000 bootstrap replicates.

    Another phylogram was constructed using one nuclear locus ITS and mitochondrial locus cox1 data from the above isolates, together with other Phytophthora species from clade 7, for a phylogenetic analysis in the same way as described above. Two isolates, P. phaseoli and P. infestans in clade 1, were used as outgroups. The unaligned regions of both ends were trimmed and evolutionary analyses were also performed in MEGA5.

    Results

    Disease symptoms.

    The diseased mung bean plants with Phytophthora stem rot symptoms exhibited dark brown lesions on basal stems. These lesions gradually expanded and finally girdled the stems, causing the plants to wilt and ultimately die (Fig. 1). The disease incidence in fields ranged from 10 to 15%.

    Fig. 1.

    Fig. 1. A and B, Dark brown lesions on basal stems of mung bean plants infected by Phytophthora vignae and C, oospores from diseased tissue observed under a microscope.

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    Pathogen isolation and morphological characterization.

    Twenty-two Phytophthora-like isolates were obtained from diseased plants (two isolates) and field soil samples (20 isolates). These isolates were homothallic. Sexual structures were produced after incubation on V8A medium for 14 days. Oogonia were spherical, produced on short stalk, and ranged in diameter from 25.1 to 41.9 µm (n = 30), with an average ± SD of 34.5 ± 2.5 µm (Fig. 2A and B). Oospores were aplerotic or nearly plerotic; their diameters ranged from 22.3 to 37.5 µm (n = 30), with an average of 30.2 ± 2.7 µm (Fig. 2A and B). Antheridia were amphigynous and cylindrical, with an average length and width of 16.9 ± 2.3 µm (10.6 to 24.1) and 15.7 ± 2.1 µm (12.3 to 20.7), respectively (n = 30; Fig. 2A and B). Sporangia were variable in shape from ellipsoid to ovoid, nonpapillate, and persistent on the stalk, with an average length and breadth of 47.2 ± 11.2 (35.1 to 67.3) × 29.4 ± 7.6 µm (20.8 to 46.2; Fig. 2C, D, E, F, and G). The sporangia were usually produced on unbranched sporangiophores that proliferated internally (Fig. 2H and I). Hyphal swellings of these isolates were formed but no chlamydospores were observed on the V8A medium. These morphological characteristics were similar to the previous description of P. vignae (Purss 1957).

    Fig. 2.

    Fig. 2. Morphological structures of the Phytophthora vignae isolates obtained from mung bean grown on V8 agar. A, Spherical oogonium with amphigynous antheridium. B, Aplerotic oospore. Sporangia produced in sterile pond water: C, E, and F, ellipsoid, nonpapillate sporangium; D, ovoid sporangia, with swollen apex before release of already-differentiated zoospores; G, release of individual zoospores; and H and I, internal nested proliferation. Scale bars = 15 μm.

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    Host range.

    Five days after inoculation, the mung bean isolates tested were pathogenic to all mung bean cultivars except that six of the 22 isolates had no pathogenicity to cultivar Sulv 3, two isolates had no pathogenicity to cultivar Zhonglv 5, and isolate PVM8 had no pathogenicity to both cultivars Sulv 3 and Zhonglv 5. The results also exhibited significant differences in virulence (P < 0.0001) among the mung bean isolates based on the statistical data analysis (i.e., ANOVA analyses and t test; Table 1; Supplementary Table S1). All mung bean isolates were also pathogenic to adzuki bean but with a lower mortality rate of seedlings than to mung bean by ANOVA analyses and t test (Table 1), indicating a relatively weak virulence to adzuki bean. There were also significant differences in virulence among mung bean isolates on each adzuki bean cultivar (P < 0.05) based on the seedling death rate. Only two of 22 mung bean isolates appeared to be strongly virulent to cultivar Suhong 2, and nine isolates had no virulence (Table 1). All inoculated susceptible plants initially exhibited constriction of stems at the wound site. As the disease developed, entire seedlings died. No symptoms were observed on the control plants or the other six crop species (cowpea, soybean, pea, chickpea, common bean, and faba bean). The inoculated pathogens were reisolated from randomly selected diseased plants but not from the controls, confirming Koch’s postulates. The other two isolates, PaV1 (P. vignae f. sp. adzukicola) and PcV2 (P. vignae f. sp. vignae), examples of two formae speciales of P. vignae, were only pathogenic toward their original hosts, adzuki bean and cowpea, respectively (Table 1).

    Phylogenetic analysis.

    BLAST analyses of the ITS, LSU, cox1, β-tubulin, EF1-α, 60SL10, Enl, HSP90, and TigA gene sequences from the 22 mung bean isolates and PaV1 and PcV2 against the NCBI GenBank database revealed that all nine loci sequences showed high similarity (up to 99 to 100%) with corresponding gene regions of many P. vignae isolates, including type strain P3019. As for the nine loci sequences, there were only a few single nucleotide polymorphism (SNP) differences in the six loci regions, including ITS (two SNPs), LSU (six SNPs), β-tubulin (11 SNPs), EF1-α (nine SNPs), 60SL10 (two SNPs), and HSP90 (two SNPs) (Table 2), and no sequence difference existed in the remaining three gene regions (cox1, Enl, and TigA). The resulting sequences of the nine gene regions for the 24 isolates (22 mung bean isolates and two P. vignae formae speciales reference isolates, PaV1 and PcV2) were trimmed and submitted to GenBank, and the accession numbers are listed in Supplementary Table S2.

    Table 2. Single nucleotide polymorphism site information in ITS, LSU, β-tubulin, EF1-α, 60SL10 and HSP90 regions between the 24 Phytophthora vignae isolates (22 mung bean isolates and two P. vignae formae speciales, PaV1 and PcV2) and P. vignae type strain P3019y

    The trimmed sequences of the eight nuclear loci were cascaded and a total sequence length of 6,476 bp from the 24 isolates and the other clade 7 species was included in the final dataset. The phylogenetic tree based on the concatenated nuclear sequence dataset of the eight different regions (ITS-LSU-β-tubulin-EF1-α-60SL10-Enl-HSP90-TigA) had a similar topology, whether maximum parsimony or maximum likelihood analyses were used (Fig. 3). The phylogenetic analysis revealed that the mung bean isolates of P. vignae were grouped together with P. vignae type strain P3019, P. vignae f. sp. vignae PcV2 (cowpea), and P. vignae f. sp. adzukicola PaV1 (adzuki bean) in subclade 7b, which also included several other Phytophthora pathogens of the legume crops such as P. sojae (soybean) and P. pisi (pea and faba bean) (Yang et al. 2017) (Fig. 3).

    Fig. 3.

    Fig. 3. Phylogenetic tree showing relationships between 24 Phytophthora vignae isolates (22 from mung bean and two from other P. vignae formae speciales, PaV1 and PcV2) and other Phytophthora species from clade 7 based on the concatenated nuclear loci internal transcribed spacer region, rRNA gene large subunit, a partial sequence of the β-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 sequences. Numbers above nodes present support values for maximum parsimony (left) and maximum likelihood (right). P. phaseoli and P. infestans in clade 1 were used as the outgroup.

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    Alignments of the nuclear locus ITS and mitochondrial locus cox1 sequences from the 24 isolates and the other clade 7 species including P. vignae type strain P3019 revealed that 83 parsimony informative sites and 28 SNPs exist in ITS, whereas 41 parsimony informative sites and 31 SNP sites exist in cox1. In addition, the 24 isolates showed a 100% sequence identity with P. vignae type strain P3019 in cox1. The phylogenetic tree constructed based on phylogenetic ITS and cox1 data showed a similar topology and all mung bean isolates clustered into one branch, together with P. vignae type strain P3019 and the reference isolates PaV1 (P. vignae f. sp. adzukicola) and PcV2 (P. vignae f. sp. vignae) (Supplementary Fig. S1).

    Discussion

    In this study, a newly emerging disease of mung bean, identified as Phytophthora stem rot, was first reported in Mingguang City, Anhui Province, and has since been observed in other regions of China, including Wuhan in Hubei Province and Nanjing in Jiangsu Province. Moreover, the disease problems have expanded and worsened. Thus, identifying and characterizing the causal pathogen of Phytophthora stem rot on mung bean is critical for establishing appropriate disease control measures.

    The Phytophthora isolates obtained from mung bean had similar morphological characteristics to those of the reported P. vignae (Purss 1957; Zhu and Wang 2003), but there was a little difference in dimensions of reproductive organs. This may be related to their respective original hosts and geographic distributions. The mung bean isolates were subsequently subjected to molecular phylogenetic analysis using sequences of nuclear and mitochondrial loci. The phylogenetic trees constructed based on the concatenated eight nuclear sequences and the combined ITS and cox1 sequences both indicated that the mung bean isolates are clustered together with PcV2 (P. vignae f. sp. vignae), PaV1 (P. vignae f. sp. adzukicola), and P. vignae type strain P3019 in a branch (Fig. 3; Supplementary Fig. S1), which further confirmed that the mung bean isolates are P. vignae. Sequence alignments of the mung bean isolates and the other two P. vignae formae speciales revealed that SNP variations exist in several gene regions, including ITS (two SNPs), LSU (six SNPs), β-tubulin (11 SNPs), EF1-α (nine SNPs), 60SL10 (two SNPs), and HSP90 (two SNPs), indicating that the mung bean P. vignae isolates are different from P. vignae f. sp. vignae and P. vignae f. sp. adzukicola.

    Host range tests in this study showed that P. vignae isolates from mung bean are pathogenic to mung bean and adzuki bean, but not to cowpea and the other five tested leguminous crops (Table 1). Moreover, the isolates exhibited stronger virulence on mung bean based on the statistical analysis. The two reference isolates used in this study, PcV2 (P. vignae f. sp. vignae) and PaV1 (P. vignae f. sp. adzukicola), were only pathogenic to their respective original hosts, cowpea and adzuki bean. The results of the pathogenicity test are consistent with previous reports by Kitazawa et al. (1978), Tsuchiya et al. (1986), and Dilantha Fernando and Linderman (1993). Kitazawa et al. (1978) revealed that the adzuki bean P. vignae isolates are only pathogenic to adzuki bean by three inoculation methods: unwounded, wounded, and soil inoculations. Tsuchiya et al. (1986) demonstrated that the adzuki bean and cowpea isolates only infect their respective original hosts using hypocotyl wound inoculation and soil inoculation. Dilantha Fernando and Linderman (1993) indicated that the cowpea isolates were only pathogenic to cowpea with soil inoculation. However, the results of pathogenicity tests performed by Zhu and Wang (2003) were different using unwounded and wounded inoculation. Zhu and Wang (2003) showed that P. vignae f. sp. adzukicola attacks only adzuki bean using unwounded inoculation but could infect mung bean and cowpea by wound inoculation. Kitazawa et al. (1979) revealed that adzuki bean and cowpea isolates of P. vignae attack both adzuki bean and cowpea using the soil inoculation method but adzuki bean isolates are more virulent to adzuki bean, whereas cowpea isolates are more virulent to cowpea. The inconsistent results of P. vignae f. sp. vignae and P. vignae f. sp. adzukicola among these studies may be caused by (i) various inoculation methods, (ii) different plant cultivars inoculated, and (iii) different geographic origins of the isolates. Combining the previous studies and our results, we can conclude that the P. vignae f. sp. adzukicola isolates are pathogenic to adzuki bean, cowpea, and mung bean and P. vignae f. sp. vignae isolates are pathogenic to cowpea and adzuki bean, whereas the mung bean P. vignae isolates are pathogenic to mung bean and adzuki bean. All results indicate that the mung bean isolates of P. vignae have a different host range from P. vignae f. sp. vignae and P. vignae f. sp. adzukicola. Thus, the P. vignae isolates obtained from mung bean are designated as P. vignae f. sp. mungcola. All of these results inspire us that further study on pathogenicity testing should be conducted on enough plant cultivars using various inoculation methods to confirm pathogenicity or virulence.

    Some species of genus Phytophthora have been divided further into races. For example, P. sojae has been reported to have >200 pathotypes/races to date (Dorrance et al. 2016). Four races have been identified in both P. vignae f. sp. vignae and P. vignae f. sp. adzukicola, respectively (Kondo et al. 2004; Notsu et al. 2003; Purss 1972; Tsuchiya et al. 1986). Analysis of the variance of mung bean isolates toward their original host revealed a significant difference in virulence level to different cultivars (Table 1). Of 22 isolates, 15 were virulent to all mung bean cultivars; PVM8 was not virulent to Sulv 3 and Zhonglv 5 and PVM9 was not virulent only to Zhonglv 5, whereas five isolates were only not virulent to mung bean cultivar Sulv 3 (Table 1), which implies that there may be four pathotypes/races among mung bean isolates. Investigations regarding pathological differentiation among mung bean isolates are in progress.

    Phytophthora disease is soilborne and the oospores of genus Phytophthora can survive for decades in soil until they are exposed to conditions that are suitable for germination. Because P. vignae causing Phytophthora stem rot has a narrow host range on legume crops, crop rotation practices, especially with nonlegume crops, may be feasible to reduce pathogen inoculum levels in soil. However, the most effective approach to control the disease is still the deployment of genetically resistant varieties. Previous studies on Phytophthora root and stem rot caused by P. sojae confirmed that a hypocotyl wound inoculation is useful for screening the disease resistance of soybean germplasms. To date, >30 resistance genes to P. sojae (Rps) have been identified in soybean based on this inoculation procedure (Zhong et al. 2019). Therefore, this method may also be applicable for identifying resistant mung bean genotypes. Moreover, because there may be more pathotypes/races among the mung bean isolates obtained in this study, the search for broad-spectrum resistant cultivars should be conducted in the greenhouse and under field conditions.

    Based on morphological and molecular characteristics, host specificity, and a multilocus phylogenetic analysis, this study identified a novel P. vignae forma specialis responsible for a new mung bean disease, Phytophthora stem rot, which was designated as P. vignae f. sp. mungcola. Our results provide a foundation for disease control and screening of resistant germplasm for breeding programs. We hope that further studies will confirm the pathotypes/races of the mung bean isolates and clarify the mechanism underlying the host specificity of three P. vignae formae speciales (P. vignae f. sp. vignae, P. vignae f. sp. adzukicola, and P. vignae f. sp. mungcola) for three leguminous crops (cowpea, adzuki bean, and mung bean).

    Acknowledgments

    We sincerely thank Prof. Benjin Li (Institute of Plant Protection, Fujian Academy of Agricultural Sciences) for providing P. vignae f. sp. vignae isolate PcV2. We thank Liwen Bianji (Edanz Group China; https://www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.

    Funding: This study was supported by grants from the Modern Agro-industry Technology Research System (CARS-08), the National Crop Germplasm Resources Center (NCGRC2020-09), and the Scientific Innovation Program of the Chinese Academy of Agricultural Sciences and the Program of Protection of Crop Germplasm Resources (2020NWB036-12) from the Ministry of Agriculture of the People’s Republic of China.