First report of anthracnose caused by Colletotrichum kahawae on Hypericum chinensis in China.

Hypericum chinensis is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019, a new leaf spot disease was observed on H. chinensis in three commercial nurseries in Kunming (25°05'N, 102°72'E), Yunnian province, China. Disease symptoms were observed on approximately 40% of the plants one year after planting and 30% of the leaves were infected. Leaf symptoms began as small, water-soaked lesions on young leaves which later became larger, dark brown and necrotic. The lesion size ranged from 0.2 to 2.8 cm in diameter. For pathogen isolation, three samples of symptomatic leaves were collected from four different nurseries. The leaves were cut into 0.5 mm pieces, surface sterilized using 70% ethanol for 30 s, and 3% NaOCl for 5 min, rinsed three times in sterilized distilled water and plated on potato dextrose agar (PDA) (Zhou et al. 2023). The plates were incubated at 26°C in the dark for 3 days. Eight isolates with comparable morphological characteristics were obtained. Initially, colonies produced pale gray to white aerial mycelia, turning dark gray after 5 days. The isolates produced hyaline, single celled, straight and cylindrical conidia, with mean size 9.7 to 14.8 µm long × 3.7 to 5.6 µm wide (n = 100). Morphological characteristics were consistent with Colletotrichum sp. (Bailey and Jeger 1992). For molecular analysis, genomic DNA was extracted from three representative isolates (XSD1, XSD3 and XSD5), amplified using the primers ITS1/ITS4 (Yin et al. 2012) and T1/Bt2b (Glass and Donaldson 1995) and submitted to sequencing (Weir et al. 2012). DNA sequences of the isolates XSD2, XSD3 and XSD8 were identical. DNA sequences of a representative isolate XSD2 were deposited in GenBank (accession no. MW202334 for ITS, and OR347007 for TUB 2). MegaBLAST analysis of the ITS and TUB2 sequences showed 99.5% and 99.3% similarity with C. kahawae strain ICMP 18539 (accession no. NR_120138.1 for ITS) and strain IMI319418 (JX145227.1 for TUB 2). Pathogenicity tests were conducted by inoculating the pathogen on healthy mature leaves of H. chinensis in the field. Ten leaves (two leaves/plant) were inoculated by spraying conidial suspension (106 spores/ml) of isolates XSD1, XSD3 and XSD5, and covered with plastic bags to maintain high humidity for 48 hours, respectively. Leaves treated with sterile distilled water served as a control. All inoculated leaves showed symptoms similar to those observed in the field at 23±5°C 10 days after inoculation. No symptoms developed on non-inoculated leaves. The pathogen was re-isolated from inoculated diseased leaves and identified as C. kahawae based on morphological and molecular characters. C. kahawae has been reported to cause leaf spot on cultivated rocket in Italy (Garibaldi et al. 2016), and anthracnose disease on tree tomato in Colombia (Rojas et al. 2018), to our knowledge, this is the first report of C. kahawae causing anthracnose on H. chinensis worldwide. Due to important ornamental and economic value of H. chinensis, the distribution of C. kahawae needs to be investigated and monitored for effective disease management strategies to be developed.

2-Oxoglutarate contributes to the effect of foliar nitrogen on enhancing drought tolerance during flowering and grain yield of soybean.

Drought severely affects the growth and yield of soybean plants especially during the flowering period. To investigate the effect of 2-oxoglutarate (2OG) in combination with foliar nitrogen (N) at flowering stage on drought resistance and seed yield of soybean under drought stress. This experiment was conducted in 2021 and 2022 on drought-resistant variety (Hefeng 50) and drought-sensitive variety (Hefeng 43) soybean plants treated with foliar N (DS + N) and 2-oxoglutarate (DS + 2OG) at flowering stage under drought stress. The results showed that drought stress at flowering stage significantly increased leaf malonaldehyde (MDA) content and reduced soybean yield per plant. However, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were significantly increased by foliar N treatment, and 2-oxoglutarate synergistically with foliar N treatment (DS + N + 2OG) was more beneficial to plant photosynthesis. 2-oxoglutarate significantly enhanced plant N content, glutamine synthetase (GS) and glutamate synthase (GOGAT) activity. Furthermore, 2-oxoglutarate increased the accumulation of proline and soluble sugars under drought stress. Under drought stress, soybean seed yield was increased by DS + N + 2OG treatment by 16.48-17.10% and 14.96-18.84% in 2021 and 2022, respectively. Thus, the combination of foliar N and 2-oxoglutarate better mitigated the adverse effects of drought stress and could better compensate for the yield loss of soybean under drought stress.

Occurrence of Geranium wilfordii anthracnose caused by Colletotrichum dematium in China.

Geranium wilfordii Maxim. is a weed of perennial herbs and considerable medicinal plant for treating acute and chronic rheumatalgia in China. In August 2019, leaf spots on G. wilfordii were observed in Harbin (45°60'N, 126°64'E), Heilongjiang Province, China. The disease occurred on 15 to 30% of G. wilfordii leaves in three nurseries (~1.5 ha/each nursery). Initial symptoms were brown necrotic spots with a gray-white center, which enlarged gradually from approximately 1 to 5 mm in diameter, and produced concentric rings and became necrotic. Twelve infected tissues from twelve diseased leaves were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water, dried on sterilized filter paper and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 26°C for 5 days. Eight fungal cultures with consistent characteristics were obtained and subcultured by transferring hyphal tips onto fresh PDA. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Colonies on PDA consisted of cottony, dense, grayish white mycelium, pale gray colony. Conidia of a representative isolate LGC2 were single-celled, hyaline, cylindrical to slightly curved with a rounded apex and truncated base that measured 16.2 to 22.5 µm (length) × 2.6 to 3.7 µm (width) (n = 50). The appressoria were elliptic to claviform or slightly lobed on synthetic nutrient-poor agar. Based on these characteristics, the eight isolates were identified as Colletotrichum dematium (Damm et al. 2009). Genomic DNA was extracted from representative isolates LGC2, LGC3, LGC5 and the internal transcribed spacer regions (ITS)，beta-tubulin (TUB2) and actin (ACT) were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), T1/Bt2b (Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. DNA sequences of isolates LGC2, LGC3, and LGC5 were identical and deposited onto the GenBank (accession nos. MW193053.1 for ITS, MZ357349.1 for TUB2, and OL956946.1 for ACT). MegaBLAST analysis showed 100%, 99.7% and 100% identical to C. dematium isolates CBS 125.25 (accession nos. NR_111453.1 for ITS 552/553 bp, GU228113.1 for TUB2 386/387 bp, and GU227917.1 for ACT 231/231 bp respectively. A pathogenicity test was performed on with a representative isolate LGC2 by spraying spore suspension (1 × 106 conidia/ml) on the surfaces of all leaves of ten healthy three-month-old G. wilfordii plants. All leaves of ten control plants were inoculated with sterile water to serve as the control. All plants were placed in a humidity chamber (>95% RH, 26℃) for 48 h after inoculation and then transfered in a greenhouse at 22/28°C with a 12:12h light-dark cycle for 10 days. All inoculated leaves showed symptoms similar to those observed in the fields, while no symptoms were observed on the control leaves. The experiment was conducted twice. The fungus was re-isolated from the infected leaves and confirmed to be C. dematium according to morphological and molecular characteristics. C. dematium has previously been reported on common knotgrass (Liu et al. 2016), on piper betle (Sun et al. 2020), peanut anthracnose in China (Yu et al. 2020). To our knowledge, this is the first report of C. dematium causing G. wilfordii anthracnose in China. G. wilfordii anthracnose caused by C. dematium poses a threat to significantly reduce the quality of G. wilfordii. Therefore, its distribution needs to be investigated and effective disease management strategies developed.

Gene expression analysis of resistant and susceptible rice cultivars to sheath blight after inoculation with Rhizoctonia solani.

BACKGROUND: Rice sheath blight, caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris), is one of the most severe diseases in rice (Oryza sativa L.) worldwide. Studies on resistance genes and resistance mechanisms of rice sheath blight have mainly focused on indica rice. Rice sheath blight is a growing threat to rice production with the increasing planting area of japonica rice in Northeast China, and it is therefore essential to explore the mechanism of sheath blight resistance in this rice subspecies. RESULTS: In this study, RNA-seq technology was used to analyse the gene expression changes of leaf sheath at 12, 24, 36, 48, and 72 h after inoculation of the resistant cultivar 'Shennong 9819' and susceptible cultivar 'Koshihikari' with R. solani. In the early stage of R. solani infection of rice leaf sheaths, the number of differentially expressed genes (DEGs) in the inoculated leaf sheaths of resistant and susceptible cultivars showed different regularity. After inoculation, the number of DEGs in the resistant cultivar fluctuated, while the number of DEGs in the susceptible cultivar increased first and then decreased. In addition, the number of DEGs in the susceptible cultivar was always higher than that in the resistant cultivar. After inoculation with R. solani, the overall transcriptome changes corresponding to multiple biological processes, molecular functions, and cell components were observed in both resistant and susceptible cultivars. These included metabolic process, stimulus response, biological regulation, catalytic activity, binding and membrane, and they were differentially regulated. The phenylalanine metabolic pathway; tropane, piperidine, and pyridine alkaloid biosynthesis pathways; and plant hormone signal transduction were significantly enriched in the early stage of inoculation of the resistant cultivar Shennong 9819, but not in the susceptible cultivar Koshihikari. This indicates that the response of the resistant cultivar Shennong 9819 to pathogen stress was faster than that of the susceptible cultivar. The expression of plant defense response marker PR1b gene, transcription factor OsWRKY30 and OsPAL1 and OsPAL6 genes that induce plant resistance were upregulated in the resistant cultivar. These data suggest that in the early stage of rice infection by R. solani, there is a pathogen-induced defence system in resistant rice cultivars, involving the expression of PR genes, key transcription factors, PAL genes, and the enrichment of defence-related pathways. CONCLUSION: The transcriptome data revealed the molecular and biochemical differences between resistant and susceptible cultivars of rice after inoculation with R. solani, indicating that resistant cultivars have an immune response mechanism in the early stage of pathogen infection. Disease resistance is related to the overexpression of PR genes, key transcriptome factors, and PAL genes, which are potential targets for crop improvement.

Genome-wide association of single nucleotide polymorphism loci and candidate genes for frogeye leaf spot (Cercospora sojina) resistance in soybean.

BACKGROUND: Frogeye leaf spot (FLS) is a destructive fungal disease that affects soybean production. The most economical and effective strategy to control FLS is the use of resistant cultivars. However, the use of a limited number of resistant loci in FLS management will be countered by the emergence of new high-virulence Cercospora sojina races. Therefore, we identified quantitative trait loci (QTL) that control resistance to FLS and identified novel resistant genes using a genome-wide association study (GWAS) on 234 Chinese soybean cultivars. RESULTS: A total of 30,890 single nucleotide polymorphism (SNP) markers were used to estimate linkage disequilibrium (LD) and population structure. The GWAS results showed four loci (p < 0.0001) distributed over chromosomes (Chr.) 5 and 20, that are significantly associated with FLS resistance. No previous studies have reported resistance loci in these regions. Subsequently, 45 genes in the two resistance-related haplotype blocks were annotated. Among them, Glyma20g31630 encoding pyruvate dehydrogenase (PDH), Glyma05g28980, which encodes mitogen-activated protein kinase 7 (MPK7), and Glyma20g31510, Glyma20g31520 encoding calcium-dependent protein kinase 4 (CDPK4) in the haplotype blocks deserves special attention. CONCLUSIONS: This study showed that GWAS can be employed as an effective strategy for identifying disease resistance traits in soybean and narrowing SNPs and candidate genes. The prediction of candidate genes in the haplotype blocks identified by disease resistance loci can provide a useful reference to study systemic disease resistance.

First report of Pythium aristosporum causing corn stalk rot in China.

Corn (Zea mays L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine pathogenic fungi causing corn stalk rot in 3 fields (~120 ha) in Harbin city (44.04°N 125.42°E), Heilongjiang Province, China. In each field, 100 plants at 5 sampling points were assessed at the milk stage (R3) of development. Disease incidence was 12%. Symptomatic plants showed rapid death of the upper leaves, drooping ears and stalks were soft, hollow, watersoaked with white hyphae present on teh outside of the stalk. Pieces of tissue (0.25 cm2) from 15 individual diseased stalks (5 plants/field) were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) containing streptomycin (50 µg/mL). After three days of incubation, a total of twelve fungal cultures with uniform characteristics were isolated and subcultured by transferring hyphal tips onto V8. Colonies on V8 selective medium were creamy white and floccus, with a growth rate of 20 mm/day at 26°C in darkness. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 µm thick (n = 50); globose oogonia, 23.9 to 30.5 µm in diameter (n = 50), and had 1 to 8 antheridia. Based on these characteristics, the isolates were identified as Pythium sp. (van der Plaats-Niterink 1981). Genomic DNA was extracted from single conidial cultures of representative isolates (MZYJF1, MZYJF3 and MZYJF7), and the internal transcribed spacer (ITS) region and cytochrome coxidase subunit II (CoxII) gene were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and COX2f/COX2r (Hudspeth et al. 2000), respectively. Partial nucleotide sequences of 796 bp and 573 bp for the ITS and COX11 amplicons, respectively, were obtained and deposited in GenBank (accession no. MW447501 for ITS, and MW471006 for COXII). MegaBLAST analysis of the ITS and CoxII sequences of MZYJF1 isolate showed 100% similarity with sequences from P. aristosporum strain ATCC 11101. The isolates were identified as P. aristosporum based on the fact that P. aristosporum has aplerotic oospores and less antheridia per oogonium than P. arrhenomanes (van der Plaats-Niterink 1981). A pathogenicity test was performed on corn cv. Xianyu 335 at tasseling stage (VT) in the field. An oospore suspension, obtained from isolate MZYJF1 grown on V8 agar media for 4 weeks (Green and Jensen, 2000) and diluted to 1×104 oospores/mL using blood cell counting method, was injected into the base of the maize stems of 6 healthy plants (1.5 ml/plant ) using a syringe. Control plants were injected with distilled sterile water. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field. The oomycete of P. aristosporum was reisolated from symptomatic plants on V8 agar media and identified according to morphological and molecular characteristics. No symptoms were observed on the control plants. P. aristosporum has previously been reported on causing damping-off of pea in the Columbia basin of Central Washington (Alcala et al. 2016) and on soybean in North Dakota (Zitnick-Anderson and Nelson 2015). To our knowledge, this is the first report of P. aristosporum causing corn stalk rot in China. Corn stalk rot caused by P. aristosporum poses a threat to significantly reduce the quality of corn. Thus, its distribution needs to be investigated and effective disease management strategies developed.

Comparative transcriptome analysis of two Cercospora sojina strains reveals differences in virulence under nitrogen starvation stress.

BACKGROUND: Cercospora sojina is a fungal pathogen that causes frogeye leaf spot in soybean-producing regions, leading to severe yield losses worldwide. It exhibits variations in virulence due to race differentiation between strains. However, the candidate virulence-related genes are unknown because the infection process is slow, making it difficult to collect transcriptome samples. RESULTS: In this study, virulence-related differentially expressed genes (DEGs) were obtained from the highly virulent Race 15 strain and mildly virulent Race1 strain under nitrogen starvation stress, which mimics the physiology of the pathogen during infection. Weighted gene co-expression network analysis (WGCNA) was then used to find co-expressed gene modules and assess the relationship between gene networks and phenotypes. Upon comparison of the transcriptomic differences in virulence between the strains, a total of 378 and 124 DEGs were upregulated, while 294 and 220 were downregulated in Race 1 and Race 15, respectively. Annotation of these DEGs revealed that many were associated with virulence differences, including scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, and ß-1,3-glucanase. In addition, two modules highly correlated with the highly virulent strain Race 15 and 36 virulence-related DEGs were found to contain mostly ß-1,4-glucanase, ß-1,4-xylanas, and cellobiose dehydrogenase. CONCLUSIONS: These important nitrogen starvation-responsive DEGs are frequently involved in the synthesis of melanin, polyphosphate storage in the vacuole, lignocellulose degradation, and cellulose degradation during fungal development and differentiation. Transcriptome analysis indicated unique gene expression patterns, providing further insight into pathogenesis.

Comparative genomics and association analysis identifies virulence genes of Cercospora sojina in soybean.

BACKGROUND: Recently, a new strain of Cercospora sojina (Race15) has been identified, which has caused the breakdown of resistance in most soybean cultivars in China. Despite this serious yield reduction, little is known about why this strain is more virulent than others. Therefore, we sequenced the Race15 genome and compared it to the Race1 genome sequence, as its virulence is significantly lower. We then re-sequenced 30 isolates of C. sojina from different regions to identifying differential virulence genes using genome-wide association analysis (GWAS). RESULTS: The 40.12-Mb Race15 genome encodes 12,607 predicated genes and contains large numbers of gene clusters that have annotations in 11 different common databases. Comparative genomics revealed that although these two genomes had a large number of homologous genes, their genome structures have evolved to introduce 245 specific genes. The most important 5 candidate virulence genes were located on Contig 3 and Contig 1 and were mainly related to the regulation of metabolic mechanisms and the biosynthesis of bioactive metabolites, thereby putatively affecting fungi self-toxicity and reducing host resistance. Our study provides insight into the genomic basis of C. sojina pathogenicity and its infection mechanism, enabling future studies of this disease. CONCLUSIONS: Via GWAS, we identified five candidate genes using three different methods, and these candidate genes are speculated to be related to metabolic mechanisms and the biosynthesis of bioactive metabolites. Meanwhile, Race15 specific genes may be linked with high virulence. The genes highly prevalent in virulent isolates should also be proposed as candidates, even though they were not found in our SNP analysis. Future work should focus on using a larger sample size to confirm and refine candidate gene identifications and should study the functional roles of these candidates, in order to investigate their potential roles in C. sojina pathogenicity.