Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Previous studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae. RESULT: This study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress. CONCLUSION: Our results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.

Interaction of negative regulator OsWD40-193 with OseEF1A1 inhibits Oryza sativa resistance to Hirschmanniella mucronata infection.

Rice is a crucial food crop worldwide, but it is highly susceptible to Hirschmanniella mucronata, a migratory parasitic nematode. No rice variety has been identified that could resist H. mucronata infection. Therefore, it is very important to study the interaction between rice and H. mucronata to breed resistant rice varieties. Here, we demonstrated that protein OsWD40-193 interacted with the extension factor OseEF1A1 and both were negative regulators inhibiting rice resistance to H. mucronata infection. Overexpression of either OsWD40-193 or OseEF1A1 led to enhance susceptibility to H. mucronata, whereas the absence of OsWD40-193 or OseEF1A1 led to resistance. Further transcriptomic analysis showed that OseEF1A1 deletion altered the expression of genes association with salicylic acid, jasmonic acid and abolic acid signaling pathways and increased the accumulation of secondary metabolites to enhance resistance in rice. Our study showed that H. mucronata infection affected the expression of negative regulators in rice and inhibited rice resistance, which was conducive to the infection of nematode. Together, our data showed that H. mucronata affected the expression of negative regulators to facilitate its infection and provided potential target genes to engineering resistance germplasm via gene editing of the negative regulators.

First Report of Colletotrichum fructicola Causing Anthracnose on Punica granatum in China.

Punica granatum L. (Pomegranate), a deciduous shrub, is widely cultivated as a fruit tree and decorative plant in China. Its flowers, leaves, roots and fruit bark also has been widely used for the treatment of different types of human disease because of the high anti-inflammatory and antibacterial activitiy (Tehranifar et al. 2011). In October 2022, leaf spot symptoms were observed on P. granatum leaves in a landscaped area on the campus of Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. A survey of 40 P. granatum of 300 m2 found that up to 20% of the foliage was infected. Infection normally starts at the tip or edge of the leaves, with the initial symptoms of lesions usually being small dark brown spots (0.8 to 1.5 mm) that gradually expand into irregular spots with grayish white central parts, and brown margins (2.3 to 3.8 mm). Ten freshly infected leaves from three different plants were collected and cut into small slices, disinfected with 75% ethanol for 30 seconds followed by 5% NaClO for 1 minute, rinsed 3 times with sterile water, and then plated on potato dextrose agar (PDA) and incubated in the dark at 25°C. After 7 days, all incubated samples produced similar morphology of aerial mycelium pale grey, dense, and cottony. Conidia were hyaline, smooth-walled, cylindrical, aseptate and measuring 12.28 to 21.05 × 3.51 to 7.37 µm (n = 50). Morphological characteristics were consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012; Park et al. 2018). For molecular identification, we used two representative isolates (HJAUP CH005 and HJAUP CH006) for genomic DNA extraction and amplification, using primers for ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b, GDF1/GDR1, ACT-512F/ACT-783R and CL1C /CL2C (Weir et al. 2012), respectively. The sequenced loci (GenBank accession nos. ITS: OQ625876, OQ625882; TUB2: OQ628072, OQ628073; GAPDH: OQ628076, OQ657985; ACT: OQ628070, OQ628071; CAL: OQ628074, OQ628075) exhibited 98 to 100% homology with corresponding sequences of C. fructicola strains (GenBank accession nos. OQ254737, MK514471, MZ133607, MZ463637, ON457800, respectively). A phylogenetic tree was constructed using the maximum-likelihood method in MEGA7.0 for the sequences of five concatenated genes (ITS-TUB2-GAPDH-ACT-CAL). Our two isolates clustered together with three strains of C. fructicola with 99% bootstrap support values in the bootstrap test (1000 replicates). The isolates were identified as C. fructicola based on morpho-molecular approach. The pathogenicity of HJAUP CH005 was tested indoors by inoculating the wounded leaves of four healthy P. granatum plants. Four leaves from each of two healthy plants were punctured with flamed needles and sprayed with a spore suspension (1 × 106 spores/ml), and four wounded leaves from each of other two plants were inoculated with mycelial plugs (5 × 5 mm3), respectively. Mock inoculations with sterile water and PDA plugs on four leaves each were used as controls. Treated plants were incubated in a greenhouse at high relative humidity, 25°C, and a photoperiod of 12 h. After 4 days, typical anthracnose symptoms similar to natural infection appeared on the inoculated leaves, whereas the control leaves remained asymptomatic. Based on morphological and molecular data, the fungus isolated from the inoculated and symptomatic leaves was identical to the original pathogen, confirming Koch's hypothesis. Anthracnose caused by C. fructicola has been reported to affect numerous plants worldwide, including cotton, coffee, grapes and citrus (Huang et al. 2021; Farr and Rossman 2023). This is the first report of C. fructicola causing anthracnose on P. granatum in China. This disease seriously affects the quality and yield of the fruit and should be of wide concern to us.

First Report of Anthracnose Caused by Colletotrichum siamense on Hydrangea macrophylla in China.

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae), a shrubby perennial plant, is widely used as an ornamental flowering plant because of its showy inflorescences and colorful sepals. In October 2022, leaf spot symptom was observed on H. macrophylla in Meiling Scenic Spot, which covers an area of about 143.58 km2 in Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). An investigation was carried out in a 500 m2 mountain area with 60 H. macrophylla plants in a residential garden, the incidence of disease observed was 28~35%. The symptoms were visible as nearly round dark brown spots on the leaves in the early stages of infection. At later stages, the spots gradually developed grayish white center with dark brown margins. To isolate the pathogen, seven leaves randomly selected from 30 infected leaves were cut into 4-mm2 pieces, surface disinfected with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and cultured at 25 °C in the dark for 7 days, and four strains with similar morphological characteristics were obtained from 7 diseased samples. Conidia were aseptate, cylindrical, hyaline, obtuse at both ends, and measured 13.31 to 17.53 × 4.43 to 7.45 µm (15.47 ± 0.83 × 5.91 ± 0.62 µm, n = 60). Morphological characteristics matched Colletotrichum siamense (Weir et al. 2012; Sharma et al. 2013). For molecular identification, two representative isolates (HJAUP CH003 and HJAUP CH004) were used for genomic DNA extraction, and the internal transcribed spacer (ITS), partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2) and partial calmodulin (CAL) were amplified, using primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R, GDF1/GDR1, Bt2a/Bt2b and CL1C/CL2C (Weir et al. 2012), respectively. The sequences were deposited in GenBank (accessions nos. ITS: OQ449415, OQ449416; ACT: OQ455197, OQ455198; GAPDH: OQ455203, OQ455204; TUB2: OQ455199, OQ455200; CAL: OQ455201, OQ455202). Concatenated sequences of the five genes were used to conduct phylogenetic analyses using the maximum-likelihood method in MEGA7.0 (Sudhir et al. 2016) and Bayesian inference analysis in MrBayes 3.2 (Ronquist et al. 2012). Our two isolates cluster together with four strains of C. siamense with 93%ML/1.00BI bootstrap support. The isolates were identified as C. siamense based on the morpho-molecular approach. Pathogenicity of HJAUP CH003 was tested indoors by inoculating detached wounded leaves of six healthy H. macrophylla plants. Three healthy plants with three leaves were punctured with flamed needles and sprayed with a 1 × 106 spores/ml spores suspension, and another three healthy plants were wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plant tissue were incubated in an artificial climate box at 25°C, 90% relative humidity and a photoperiod of 12 h. After 4 days, symptoms similar to those of natural infection were observed on all wounded inoculated leaves, while no symptoms appeared on mock-inoculated leaves. The fungus isolated from inoculated leaves was identical to the original pathogen based on morphological and molecular data, confirming Koch's hypothesis. It has been reported that C. siamense can cause anthracnose on numerous plants (Rong et al. 2021; Tang et al. 2021; Farr and Rossman 2023). This is the first report of C. siamense causing anthracnose on H. macrophylla in China. The disease is of major concern to the horticultural community as it seriously affects the aesthetic value of ornamentals.

Inhibition of rice germination by ustiloxin A involves alteration in carbon metabolism and amino acid utilization.

Ustiloxins are the main mycotoxin in rice false smut, a devastating disease caused by Ustilaginoidea virens. A typical phytotoxicity of ustiloxins is strong inhibition of seed germination, but the physiological mechanism is not clear. Here, we show that the inhibition of rice germination by ustiloxin A (UA) is dose-dependent. The sugar availability in UA-treated embryo was lower while the starch residue in endosperm was higher. The transcripts and metabolites responsive to typical UA treatment were investigated. The expression of several SWEET genes responsible for sugar transport in embryo was down-regulated by UA. Glycolysis and pentose phosphate processes in embryo were transcriptionally repressed. Most of the amino acids detected in endosperm and embryo were variously decreased. Ribosomal RNAs for growth were inhibited while the secondary metabolite salicylic acid was also decreased under UA. Hence, we propose that the inhibition of seed germination by UA involves the block of sugar transport from endosperm to embryo, leading to altered carbon metabolism and amino acid utilization in rice plants. Our analysis provides a framework for understanding of the molecular mechanisms of ustiloxins on rice growth and in pathogen infection.

Integrated transcriptome and metabolome analysis revealed that flavonoids enhanced the resistance of Oryza sativa against Meloidogyne graminicola.

Rice is a crucial food crop worldwide, but its yield and quality are significantly affected by Meloidogyne graminicola is a root knot nematode. No rice variety is entirely immune to this nematode disease in agricultural production. Thus, the fundamental strategy to combat this disease is to utilize rice resistance genes. In this study, we conducted transcriptome and metabolome analyses on two rice varieties, ZH11 and IR64. The results indicated that ZH11 showed stronger resistance than IR64. Transcriptome analysis revealed that the change in gene expression in ZH11 was more substantial than that in IR64 after M. graminicola infection. Moreover, GO and KEGG enrichment analysis of the upregulated genes in ZH11 showed that they were primarily associated with rice cell wall construction, carbohydrate metabolism, and secondary metabolism relating to disease resistance, which effectively enhanced the resistance of ZH11. However, in rice IR64, the number of genes enriched in disease resistance pathways was significantly lower than that in ZH11, which further explained susceptibility to IR64. Metabolome analysis revealed that the metabolites detected in ZH11 were enriched in flavonoid metabolism and the pentose phosphate pathway, compared to IR64, after M. graminicola infection. The comprehensive analysis of transcriptome and metabolome data indicated that flavonoid metabolism plays a crucial role in rice resistance to M. graminicola infection. The content of kaempferin, apigenin, and quercetin in ZH11 significantly increased after M. graminicola infection, and the expression of genes involved in the synthetic pathway of flavonoids also significantly increased in ZH11. Our study provides theoretical guidance for the precise analysis of rice resistance and disease resistance breeding in further research.

Morphological and Phylogenetic Analyses Reveal Three New Species of Distoseptispora (Distoseptisporaceae, Distoseptisporales) from Yunnan, China.

Three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are described and illustrated from specimens collected on dead branches of unidentified plants in Yunnan Province, China. Phylogenetic analyses of LSU, ITS, and TEF1 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), reveal the taxonomic placement of D. mengsongensis, D. nabanheensis, and D. sinensis within Distoseptispora. Both morphological observations and molecular phylogenetic analyses supported D. mengsongensis, D. nabanheensis, and D. sinensis as three new taxa. To extend our knowledge of the diversity of Distoseptispora-like taxa, a list of recognized species of Distoseptispora with major morphological features, habitat, host, and locality is also provided.

First Report of Corynespora cassiicola Causing Leaf Spot on Jasminum nudiflorum in China.

Winter jasmine (Jasminum nudiflorum Lindl.), a trailing, deciduous shrub, is widely used as an ornamental plant. Its flowers and leaves also has great medicinal value for treatment of inflammatory swelling, purulent eruptions, bruises and traumatic bleeding (Takenaka et al. 2002). In October 2022, leaf spot symptoms were observed on J. nudiflorum distributed in Meiling Scenic Spot (28.78°N, 115.83°E) and Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. In a week-long series of investigations, the incidences of disease could range up to 25%. Initially, the symptoms of the lesions were small yellow circular spots (0.5 to 1.8 mm), and gradually developing irregular spots (2.8 to 4.0 mm) with grayish white central parts, a dark brown inner ring, and outer yellow halo. To identify the pathogen, sixty symptomatic leaves from fifteen different plants were collected, of which twelve were randomly selected, cut into 4-mm2 pieces, and surface sterilized with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed four times with sterile water, and then placed on potato dextrose agar (PDA) medium at 25 °C in the dark for 5 to 7 days. Six isolates with similar morphological characteristics were obtained. Aerial mycelium was vigorous, downy and exhibited white to grayish-green coloration. Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse, one to 11 pseudosepta, 24.9 to 125.7 × 7.9 to 12.9 µm (n = 50). Morphological characteristics matched Corynespora cassiicola (Ellis 1971). For molecular identification, two representative isolates (HJAUP C001 and HJAUP C002) were selected for genomic DNA extraction, and the ITS, TUB2 and TEF1-α gene were amplified, using the primer ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Lousie and Donaldson 1995) and EF1-728F/EF-986R (Carbone and Kohn 1999), respectively. The sequenced loci (GenBank accession nos. ITS: OP957070, OP957065; TUB2: OP981639, OP981640; TEF1-α: OP981637, OP981638) of the isolates were 100, 99 and 98% similar to the corresponding sequences of C. cassiicola strains (GenBank accession nos. OP593304, MW961419, MW961421, respectively). Phylogenetic analyses of combined ITS and TEF1-α sequences was performed using maximum-likelihood method in MEGA 7.0 (Kuma et al. 2016). The result showed that our isolates (HJAUP C001 and HJAUP C002) clustered with four strains of C. cassiicola at 99% bootstrap values in the bootstrap test (1000 replicates). Based on the morpho-molecular approach, the isolates were identified as C. cassiicola. The pathogenicity of one representative strain (HJAUP C001) was tested by inoculating the wounded leaves of six healthy J. nudiflorum plants under natural condition. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three wounded leaves from each of other three plants were inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each, respectively. Leaves from all treatments were incubated in a greenhouse at high relative humidity, 25°C, and 12-hour photoperiod. After one week, all wounded inoculated leaves appeared similar symptoms as described above, whereas the mock inoculated leaves were still healthy. Similar isolates with grayish white and vigorous aerial mycelium were reisolated from inoculated and symptomatic leaves and identified as C. cassiicola by DNA sequencing, fulfilling Koch's postulates. It has been reported that C. cassiicola can cause leaf spots on numerous plant species (Tsai et al. 2015; Lu et al. 2019; Farr and Crossman 2023). However, to our knowledge, this is the first report of C. cassiicola causing leaf spots on J. nudiflorum in China. This finding aids in protection of J. nudiflorum, a medicinal and ornamental plant with high economic value.

Molecular Phylogeny and Morphology Reveal Four Novel Species of Corynespora and Kirschsteiniothelia (Dothideomycetes, Ascomycota) from China: A Checklist for Corynespora Reported Worldwide.

Plant debris are habitats favoring survival and multiplication of various microbial species. During continuing mycological surveys of saprobic microfungi from plant debris in Yunnan Province, China, several Corynespora-like and Dendryphiopsis-like isolates were collected from dead branches of unidentified perennial dicotyledonous plants. Four barcodes, i.e., ITS, LSU, SSU and tef1-α, were amplified and sequenced. Morphological studies and multigene phylogenetic analyses by maximum likelihood and Bayesian inference revealed three new Corynespora species (C. mengsongensis sp. nov., C. nabanheensis sp. nov. and C. yunnanensis sp. nov.) and a new Kirschsteiniothelia species (K. nabanheensis sp. nov.) within Dothideomycetes, Ascomycota. A list of identified and accepted species of Corynespora with major morphological features, host information and locality was compiled. This work improves the knowledge of species diversity of Corynespora and Kirschsteiniothelia in Yunnan Province, China.

First Report of Colletotrichum siamense Causing Anthracnose on Eriobotrya japonica in China.

Eriobotrya japonica (Thunb.) Lindl. is a subtropical evergreen tree with economic and medicinal value. In 2021-2022, leaf-spot symptoms were observed on the leaves of E. japonica in Nanchang city, Jiangxi Province, China (28°68'N, 115°95'E). The disease incidence was 30% (20 diseased plants/60 surveyed plants). Symptoms included brown spots that gradually turned dark brown. The lesions were ca. 3-8 mm, and coalescing into irregular or round large lesions. Black acervuli were observed within the lesions. The margin of the diseased tissues was cut and surface sterilized in 75% ethanol for 10 s and 0.1% (v/v) mercuric chloride for 1 min, followed by three rinses in sterile water. Thirteen single spore isolates were purified and deposited in the Mycological Herbarium of Jiangxi Agricultural University. After 7 days, the colonies were grey-white with dense aerial mycelium. Conidia were uni-celled, hyaline and cylindrical. The sizes of the conidia were 12.6 to 17.5 × 4.2 to 6.5 µm. Appressoria were oval to irregular in shape and dark brown in color. These characteristics were consistent with descriptions of Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde (Weir et al. 2012; Rodríguez-Palafox et al. 2021). The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and actin (ACT) genes were amplified and sequenced (Diao et al. 2017). The sequences were submitted to GenBank with accession numbers ON631874, ON642546, ON642547 and ON642548, respectively. BLASTn searches confirmed high identity (>99%) with the type-strain of C. siamense (MH863513, KC297007, JX009702, JX009549). The concatenated sequences were used to construct a phylogenetic tree. The present isolate JXCS1 formed a single clade with the C. siamense. For pathogenicity, the leaves of two-year-old seedlings (cv. Dawuxing) were inoculated with 10 µL of spore suspension (1×106 conidia/mL). Leaves inoculated with sterile distilled water served as controls. Each treatment was replicated three times. Five days post-inoculation, water-soaked lesions appeared on the leaves, lesions gradually expanded into large round necrotic spots. No symptoms were observed on the control plants. C. siamense was reisolated from all inoculated samples, fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on E. japonica in China. The results further expand the range of plants that can be infected by C. siamense. This disease may decrease the value of plants and proper management strategies should be applied.

Transcriptome Analysis of Protein Kinase MoCK2, which Affects Acetyl-CoA Metabolism and Import of CK2-Interacting Mitochondrial Proteins into Mitochondria in the Rice Blast Fungus Magnaporthe oryzae.

The rice pathogen Magnaporthe oryzae causes severe losses to rice production. Previous studies have shown that the protein kinase MoCK2 is essential for pathogenesis, and this ubiquitous eukaryotic protein kinase might affect several processes in the fungus that are needed for infection. To better understand which cellular processes are affected by MoCK2 activity, we performed a detailed transcriptome sequencing analysis of deletions of the MoCK2 b1 and b2 components in relation to the background strain Ku80 and connected this analysis with the abundance of substrates for proteins in a previous pulldown of the essential CKa subunit of CK2 to estimate the effects on proteins directly interacting with CK2. The results showed that MoCK2 seriously affected carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, and the related transporters and reduced acetyl-CoA production. CK2 phosphorylation can affect the folding of proteins and especially the effective formation of protein complexes by intrinsically disordered or mitochondrial import by destabilizing soluble alpha helices. The upregulated genes found in the pulldown of the b1 and b2 mutants indicate that proteins directly interacting with CK2 are compensatorily upregulated depending on their pulldown. A similar correlation was found for mitochondrial proteins. Taken together, the classes of proteins and the changes in regulation in the b1 and b2 mutants suggest that CK2 has a central role in mitochondrial metabolism, secondary metabolism, and reactive oxygen species (ROS) resistance, in addition to its previously suggested role in the formation of new ribosomes, all of which are processes central to efficient nonself responses as innate immunity. IMPORTANCE The protein kinase CK2 is highly expressed and essential for plants, animals, and fungi, affecting fatty acid-related metabolism. In addition, it directly affects the import of essential mitochondrial proteins into mitochondria. These effects mean that CK2 is essential for lipid metabolism and mitochondrial function and, as shown previously, is crucial for making new translation machinery proteins. Taken together, our new results combined with previously reported results indicate that CK2 is an essential protein necessary for the capacities to launch efficient innate immunity responses and withstand the negative effects of such responses necessary for general resistance against invading bacteria and viruses as well as to interact with plants, withstand plant immunity responses, and kill plant cells.

First report of spotting disease caused by Fusarium proliferatum infection of Alisma orientale in China.

Alisma orientale (Alismatidae) is highly valued for both its pharmaceutical and nutritional properties. The tubers are in Chinese herbal medicine and the leaves and stems for several Chinese delicacies. Intercropping A. orientale and Nelumbo nucifera may increase quality, yield, and other economic benefits. In July 2021, a novel spotting disease was observed in these plants in the White Lotus Science and Technology Expo Park in Guangchang County, Fuzhou City, Jiangxi Province (26.79°N, 116.31°E). The symptom was round to regular black spots on the stems during the early stage of infection. Over time, the larger spots merged, resulting in stem breakage and eventually death. A. orientale spot disease arose in July of 2021, causing approximately 50% of leaves to die, and leading to 10 to 25% yield loss. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for seven days, aerial mycelia were identified. At the start of culture, the mycelia were white but later turned purple-red. Three to five straight or partially bent septa were visible on the macroconidia, which were 28.8(19.1~38.6) ×2.9(1.9~4.0) µm in size (n=50). In contrast, the microconidia appeared glassy and elliptical, with sizes of 9.8(4.9~14.8) ×2.7(1.2~4.1) µm (n=50). These features suggested F. proliferatum (Zhao et al., 2019). To verify this, various primers, including universal ITS1/ITS4, Fusarium-specific EF1T/EF2T, PRO1/PRO2 (Mulè et al., 2004), and Bt2a/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1998) primers were used for amplification of the 5.8S rRNA/ITS, α-elongation factor, calmodulin, and ß-tubulin genes. The resulting sequences were between 99% and 100% identical to those of F. proliferatum in GenBank (accession numbers MW721116.1, KR071735.1, KU604008.1, and MH398186.1, respectively). The present sequences were uploaded with accession numbers of OK047496, OL448294, OL448295, and OM280358, with sequence lengths of 549 bp, 725 bp, 594 bp, and 325bp, respectively. A maximum likelihood-phylogenetic tree was created in MEGA5 based on ITS+TEF+PRO sequences. Pathogenicity was tested by hyphal inoculation. Needles, cotton, and water were sterilized under high temperature and pressure. Five-millimeter punches were taken from infected and uninfected PDA plates and three uninfected stems of A. orientale were inoculated with the pathogen with a fourth used as the control. Plants were maintained in experimental field of the Bailian Science and Technology Expo Park. Infected wounds were gently wetted with sterile water and sealed with sellotape. After 10 days, the infected stems displayed symptoms while the controls did not. The same pathogen was recovered from the infected stems, fulfilling Koch's requirements. This appears to be the only report describing F. proliferatum infection of A. orientale stems. These results are useful for the recognition and avoidance of F. proliferatum infections in A. orientale and other plants.

Genome and Transcriptome Sequencing Analysis of Fusarium commune Provides Insights into the Pathogenic Mechanisms of the Lotus Rhizome Rot.

Fusarium wilt, a vascular wilt caused by F. commune, has been a serious problem for the lotus. Although some F. commune isolate genomes have been sequenced, little is known about the genomic information of the strain that causes Fusarium wilt of aquatic plants. In this study, the genome of F. commune FCN23 isolated from lotuses in China was sequenced using Illumina and PacBio sequencing platforms. The FCN23 genome consisted of 53 scaffolds with a combined size of 46,211,149 bp. According to the reference genome, F. oxysporum f. sp. lycopersici 4287 isolated from tomato, it was finally assembled into 14 putative chromosomes, including 10 core and 4 lineage-specific chromosomes. The genome contains about 3.45% repeats and encodes 14,698 putative protein-coding genes. Among these, 1,038 and 296 proteins were potentially secreted proteins and candidate effector proteins, respectively. Comparative genomic analysis showed that the CAZyme-coding genes and secondary metabolite biosynthesis genes of FCN23 were similar to those of other Ascomycetes. Additionally, the transcriptome of FCN23 during infection of lotus was analyzed and 7,013 differentially expressed genes were identified. Eight putative effectors that were upregulated in the infection stage were cloned. Among them, F23a002499 exhibited strong hypersensitive response after transiently expressed in Nicotiana benthamiana leaves. Our results provide a valuable genetic basis for understanding the molecular mechanism of the interaction between F. commune and aquatic plants. IMPORTANCE Fusarium commune is an important soilborne pathogen with a wide range of hosts and can cause Fusarium wilt of land plants. However, there are few studies on Fusarium wilt of aquatic plants. Lotus rhizome rot mainly caused by F. commune is a devastating disease that causes extensive yield and quality losses in China. Here, we obtained high-quality genomic information of the FCN23 using Illumina NovaSeq and the third-generation sequencing technology PacBio Sequel II. Compared to the reference genome F. oxysporum f. sp. lycopersici strain 4287, it contains 11 core and 3 lineage-specific chromosomes. Many differentially expressed genes associated with pathogenicity were identified by RNA sequencing. The genome and transcriptome sequences of FCN23 will provide important genomic information and insights into the infection mechanisms of F. commune on aquatic plants.

Leaf vein disease in Nelumbo nucifera caused by Nigrospora pyriformis infection in China.

Nelumbo nucifera (Nymphaeaceae) has both ornamental and nutritional uses in China. In July 2021, a novel disease was observed on plants in the White Lotus Science and Technology Expo Park in Guangchang county, Fuzhou city, Jiangxi province (26.79° N, 116.31° E). Infection was visible as nearly round black spots on the leaves in the early stages of infection. At later stages, the spots spread along the veins forming reticular necrosis until the entire leaf was infected, and the disease caused approximately 20% of leaves to die. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for five days, white regularly shaped round colonies were visible that after 10 days turned black with fluffy hyphae. The individual conidia produced by the conidiophore were initially a light-brown color which changed to black as the pigment accumulated; conidia were globose to subglobose, 17.10 (14.77-21.66) ×14.70 (12.08-16.93) µm in size (n=50), with glossy even surfaces lacking in septa, suggestive of Nigrospora. To verify this, PCR amplification was conducted using ITS1/ITS4 primers for the 5.8S rRNA gene. The primers EF1-728F and EF-2 were used for amplifying translation elongation factor 1α (O'Donnell et al. 1998, Carbone and Kohn 1999), and Bt-2a and Bt-2b (Glass and Donaldson 1995) for amplifying ß-tubulin. The sequences were found to be 99% to 100% identical to those of Nigrospora pyriformis in GenBank (accession numbers NR153469.1, KY019290.1, and KY019457.1, respectively). The sequences were uploaded to GenBank (accession numbers OK605048, OL362197, and OK662966), with lengths of 522 bp, 475 bp, and 410 bp, respectively. A maximum-likelihood phylogenetic tree was created in MEGA5. Pathogenicity was tested by four isolates hyphal inoculation and conducted in an experimental field of the White Lotus Science and Technology Expo Park. Five-millimeter discs were taken from infected and uninfected PDA plates and inoculated into five 1-month-old healthe lotus leaves using inoculations, three with the pathogen and an uninfected sample as the control every leaf. The discs were covered with moisturized sterile cotton and fixed with transparent tape. The wounds were moistened with sterile water and sealed with adhesive tape. After 14 days, spots were visible at the infected sites while the control sites showed no symptoms. The same pathogen was recovered from the infected leaves, fulfilling Koch's requirements. Leaf spot diseases resulting from N. pyriformis infection or infection with other Nigrospora species include infection of Chenopodium album by N. pyriformis (Chen et al. 2020), Eclipta prostrata by N. sphaerica (Qiu et al. 2022), Nicotiana tabacum by N. oryzae (Wang et al. 2022), and Oxalis corymbosa by N. hainanensis (Zheng et al. 2021). This investigation appears to be the first identification of Nelumbo nucifera vein disease resulting from Nigrospora pyriformis infection. The present results are useful for the management and avoidance of the disease caused by Nigrospora pyriformis.

First report of Alternaria alternata causing leaf spot on Nelumbo nucifera plants in China.

White lotus (Nelumbo nucifera) is an aquatic plant of the Nymphaeaceae family that primarily serves as an ornamental plant and is an important cash crop in China. In May 2020, an unknown leaf disease affecting these plants was first detected in White Lotus Science and Technology Expo Park in Guangchang County, Fuzhou City, Jiangxi Province (26.79° N, 116.31° E). The disease caused approximately 30-40% of leaves to die, and led to 15 to 20% in seed yield losses. This disease was characterized by the formation of irregular yellow-brown to dark-brown spots during the initial phases of infection. As the disease is developing, these spots expanded until they were generally round and brown to purple-brown in color, with a yellow halo surrounding the expanding spots. In an effort to characterize the causative pathogen, a small ~5×5 mm leaf tissue section from the boundary between normal and diseased tissue was collected, and sterilized with the following regimes: 30 s with 75% ethanol, soaked in 0.1% mercuric chloride for 30 s, washed thrice with sterile water, and transferred onto potato dextrose agar (PDA) plate, and placed in an illumination incubator (12 h light/dark) at 28 °C± 1°C for 5 days. Seven pure cultures were obtained from ten diseased leaves. For pathogenicity testing, a hyphal inoculation strategy was employed, with all studies being conducted at the Plant Pathology Laboratory of Jiangxi Agricultural University. Five mm discs were selected from three separate cultures and one control (PDA). Healthy leaves of lotus seeds were treated with 4 treatments per leaf including three separate cultures and one control that were treated with the test pathogen. The experiments were repeated three times with three biological replicates. Healthy leaves were covered with moisturized sterile cotton balls and fixed to the leaf surface with transparent tape. The inoculated lotus seedlings were kept in greenhouse incubator at 28 °C± 3°C and relative humidity of 70 to 80%. Following a 14-day incubation period, brown spots began to manifest at all sites inoculated with the test pathogen whereas the control spots remained healthy. Diseased spots were then separated. The same pathogen was once again successfully isolated and identified microscopically, thus fulfilling Koch's postulates. Six isolates were characterized. Ovoid or elliptical conidia were brown to light-brown in color with a short beak, 1-5 diaphragms, and 0-3 mediastinum. The diameter of these conidia were thick (13.8-44.0×7.5-16.3 µm; average: 24.0×11.9 µm, n=50). These morphological characteristics were consistent with Alternaria alternata. The ITS4/ITS5, EF1-728F/EF1-986R, AltF/AltR, OPA10-2R/OPA10-2L, EPGF/EPGR and KOG1058F2/KOG1058R2 primer sets were then used to conduct molecular identification by amplifying key transcription elongation factor and internal transcriptional spacer regions, yielding sequences that were 99%-100% similar to Alternaria alternata (GenBank No: MK396606, MT178329, MN184998, MN894688, MT849815 and KP125234). Sequences were deposited in GenBank with accession numbers MW898580 (ITS, 620 bp), MW981281 (EF1-α, 284 bp), MZ514094 (Alt a1, 477 bp), MZ514095 (OPA10-2, 716 bp), MZ514096 (endoPG, 465 bp) and MZ514097 (KOG1058, 877 bp). Nelumbo nucifera is an important aquatic cash crop in China, and this is the first study we are aware of demonstrating the presence of a leaf disease caused by Alternaria alternata in Nelumbo nucifera plants anywhere in the world. These findings may offer a foundation for efforts to prevent diseases caused by Alternaria alternata.

First Report of Lotus Seedpod Withering Caused by Fusarium proliferatum on Nelumbo nucifera Plants in China.

Nelumbo nucifera (Nymphaeaceae family) is a well-known plant in China and with the increasing value of this crop, the planting area of lotus is expanding. In May 2019, an unknown withering lotus seedpod was obtained in Guangchang County of Jiangxi Province (26.79°N, 116.31°E). The disease arose between May and July of each year, resulted in the withering and consequent death of ~10% of lotus seedpods, with the disease being most serious during the rainy season. The initial symptoms of this disease include the shrinking of young lotus seedpods with concomitant yellowing of the epidermal tissue layer. These pods failed to grow normally and could to wither and die within one week, with the withering symptoms gradually spreading to associated stem tissues. To characterize the pathogens responsible for this disease, ten diseases seedpods were collected and cut into pieces of ~5×5 mm, then sterilized with 75% ethanol for 30 s, and treated with 0.1% mercuric chloride for 5 min. After being washed four times under sterilized water, samples were then transferred onto potato dextrose agar (PDA) and incubated for 7 d at 28℃ in the dark. Eight purified isolates yielded large numbers of aerial mycelium that were initially white in color, but then changed to a purple-red color over the course of this incubation period. The average mycelial growth rate was 6.3 mm per day (n=5). On PDA, macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The microconidia were hyaline, ovoid or ellipse and 4.6-13.5×2.2-4.3 µm in size (average: 8.7×3.1 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). To confirm the identity of this putative pathogen at the molecular level, the universal ITS4/ITS5 primers (White et al., 1990), the Fusarium specific pair PRO1/PRO2 (Mulè et al., 2004), EF1T/EF2T (O'Donnell et a., 1998) and RPB2F/R (O'Donnell et al., 2010) primers were utilized to amplify the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-internal transcribed spacer 2 (ITS2), calmodulin, alpha elongation factor genes, and RNA-dependent DNA polymerase II subunit from these isolates. Following alignment of the resultant sequences with GenBank via a BLAST analysis, the sequences (GenBank accession numbers: MW862499, MW762531, MW767988, MW831311, respectively.) showed 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank accession numbers: MW817705, LS423443, MH153750, and MW091308, respectively.). Based upon these morphological and molecular findings, this pathogen was identified as F. proliferatum. Pathogenicity testing was then performed using five plump healthy lotus seedpods. Sterile needles were used to generate wounds (2 mm deep, 1 mm in diameter) a 10 µL suspension of prepared spores (1.0×106 spores/mL) derived from a 7-day-old culture grown on PDA was injected into the wound sites of the lotus seedpod. As a control, give seedpods were additionally wounded and injected as the same as treated with 10 µL of sterile water. The experiments were repeated three times with five biological replicates. All seedpods were then incubated at 28℃ in a growth chamber (12 h light/dark) with 80% relative humidity. After a 3-day incubation period, wounded sites injected with spore suspensions exhibited browning. Following a 5-day incubation period, a mean lesion diameter of 9.8 mm was observed, with white mycelia growing on the wound surface and with evident withering of the internal and external tissues near the wounded site. In contrast, blank control wound sites remained healthy. We were again able to isolate F. proliferatum from the infected lotus seedpods. Finally, eight isolates were obtained were identified as the pathogen based on these morphological and molecular analyses, thus fulfilling Koch's postulates. This is the first report to our knowledge to have described a case of F. proliferatum causing lotus seedpod withering in China, providing a foundation for future research efforts aimed at presenting diseases caused by this pathogen.

Chemical components from the stems and leaves of Clausena lansium (Lour.) Skeels and their potential herbicidal effects.

BACKGROUND: Clausena lansium (Lour.) Skeels, belonging to the genus Clausena of the family Rutaceae, has a wide range of medical and agricultural activities. Previous studies on agricultural activities have shown that C. lansium extracts and some components have obvious herbicidal activities. In order to study systematically herbicidal activity of this plant, we studied the herbicidal effect of ethyl acetate (EtOAc) extract from the stems and leaves of this plant and further isolated the active compounds. RESULTS: The EtOAc extract inhibited the growth of roots and shoots of Echinochloa crus-galli (L.) Beauv., and the inhibitory effect of the EtOAc extract on roots were stronger than those on shoots with half maximal inhibitory concentration (IC50 ) values of 420.45 and 585.05 mg L-1 , respectively. Fifteen compounds were subsequently isolated and identified from the stems and leaves of C. lansium, including nine O-monoterpenoid furanocoumarins and six cinnamamides. Our results showed that most compounds exhibited varying degrees of herbicidal activities to E. crus-galli. Among them, compounds 3, 8, and 13-15 showed the best inhibitory activities on the growth of E. crus-galli roots, with inhibition rate values ranging from 70% to 83% at a concentration of 300 mg L-1 . Compounds 1 and 2 are two new compounds, and their structures were established as 5-O-monoterpenoid furanocoumarin and 8-O-monoterpenoid furanocoumarin, and named as claulansicoumarin-A and -B, respectively. CONCLUSION: The EtOAc extract and pure compounds showed noticeable herbicidal activities against E. crus-galli and indicated a great potential for these natural compounds to be developed as a herbicide. © 2020 Society of Chemical Industry.

Transcriptome analysis of roots from resistant and susceptible rice varieties infected with Hirschmanniella mucronata.

Hirschmanniella mucronata is a plant-parasitic nematode that is widespread in rice production areas and causes 10-25% yield losses a year on average. Here, we investigated the mechanism of resistance to this nematode by comparing the transcriptomes of roots from resistant (Jiabali) and susceptible (Bawangbian) varieties of rice. Of 39 233 unigenes, 2243. exhibited altered total expression levels between control and infected resistant and susceptible varieties. Significant differences were observed in the expression levels of genes related to stress, peptidase regulation or inhibition, oxidoreductase activity, peroxidase activity and antioxidant activity. The up-regulated genes related to plant secondary metabolites, such as phenylpropanoid, lignin, cellulose or hemicellulose, may result in an increase in the degree of resistance of Jiabali to the H. mucronata infection compared with that of Bawangbian by affecting cell wall organization or biogenesis. Of the genes that responded similarly to H. mucronata infection, ~252 (~76.59%) showed greater changes (whether induced or suppressed) in RN155 (susceptible varieties infected by rice root nematode) than in RN51 (resistance varieties infected by rice root nematode). Nineteen pathogenesis-related genes belonging to nine pathogenesis-related gene families were significantly induced by H. mucronata in the infected roots of Jiabali and Bawangbian, and 13 differentially expressed genes showed changes in their abundance only in the susceptible Bawangbian variety. This study may help enhance our understanding of the mechanisms underlying plant resistance to nematodes.

The complete mitogenome of Fusarium equiseti.

The complete mitochondrial genome of plant pathogenic fungus, Fusarium equiseti, was sequenced. The circular molecule is 53,411 bp long with a GC content of 32.81%. It contains 22 protein-coding genes, 4 ribosomal RNA (rRNA), and 24 transfer RNA (tRNA) genes. Phylogenetic reconstructions confirmed that it has the closest relationship with Fusarium equiseti. The mitogenome analysis of Fusarium equiseti provides a molecular basis for further studies on molecular systematics and evolutionary dynamics.