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.

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.

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.

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.

Endophytic bacterial community diversity in two citrus cultivars with different citrus canker disease resistance.

The selective infection of Xanthomonas citri pv. citri (Xcc) to citrus cultivars is universally known, but the relationship between endophytic bacteria and the resistance of host variety to canker disease remains unclear. In this study, endophytic bacterial populations of two citrus cultivars-the resistant satsuma mandarin and the susceptible Newhall navel orange-were analyzed through high-throughput sequencing. The results showed that endophytic bacterial community of satsuma mandarin was more abundant than that of Newhall navel orange. In addition, bacterial abundance was the highest in the spring samples, followed by that in summer and winter samples, in both the varieties. In all samples, the predominant phyla were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes; the major genera were Bacillus and Stenotrophomonas, and the main species was Bacillus subtilis. According to the analysis of the predominant bacteria in the two citrus cultivars, B. subtilis with potential antagonistic characteristics against Xcc existed universally in all samples. However, the susceptible Newhall navel oranges were abundant in Bacillus subtilis and had a relatively large number of canker-causing cooperative bacteria such as Stenotrophomonas. The results suggested that endophytic bacterial community of the two citrus cultivars had some differences based on the season or plant tissue, and these differences were mainly in the quantity of bacteria, affecting citrus canker disease occurrence. In conclusion, the differences in endophytic bacteria on citrus cultivars might be related to host resistance or susceptibility to citrus canker disease.

First Report of Pestalotiopsis chamaeropis Causing Leaf Spot on Eurya nitida in China.

Eurya nitida Korth. belonging to the family Theaceae is an evergreen shrub or small tree and is usually used as a very important ornamental tree and nectar source plant (Khan et al. 1992; Ma et al. 2013). It also has high medicinal values with the treatment of rheumatoid arthritis, diarrhea, innominate inflammatory of unknown origin, ulcer fester and traumatic hemorrhage (Park et al. 2004). In October 2020, symptoms of leaf spot were observed on E. nitida in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 300 m2 of the mountain area which holds about 100 trees of E. nitida scattered naturally near the waterside or regularly planted on either side of the mountain road. Most of the infected plants were observed from humid environments or waterside, with 15~20% disease incidence, and the disease severity on a plant basis was determined to be 25% to 30%, depending on the field. Sixty infected leaves were collected from 20 individual trees which have the same symptoms. The symptoms on infected leaves appeared as tiny circular spots that gradually enlarged into brown circular necrotic lesions and then became a light gray with brown borders and black acervuli at the later stages of the disease. Ten leaves of infected tissues randomly selected from collected sixty infected leaves were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Colonies developed copious white aerial mycelium covering the entire Petri dish area after 7 to 10 days. Conidiogenous cells were discrete, hyaline, and smooth. Conidia were fusiform, ellipsoid, 4-euseptate and ranged from 21.86 to 29.80 × 5.95 to 9.80 µm. Apical cells were hyaline with 2 to 3 unbranched, tubular apical appendages (mostly 3); basal cell was hyaline, obconic with a truncate base; three median cells doliiform to subcylindrical, brown. The morphological characteristics of all isolates matched features described for Pestalotiopsis chamaeropis Maharachch., K.D. Hyde & Crous (Maharachchikumbura et al. 2014). Two single representatives (JAUCC L001-1 and JAUCC L002) were used for molecular identification, which were verified based on the amplification of DNA sequences of internal transcribed spacer region (ITS) gene and translation elongation factor 1 alpha (TEF1-α) gene, using the primers ITS4/ITS5 (White et al. 1990) and EF1-526F/EF1-1567R (Rehner and Buckley 2005), respectively. The sequenced loci (GenBank accession nos. ITS: MW845761, MW828589 and TEF1-α: MW838967, MZ292464) exhibited over 99% homology with P. chamaeropis strain CBS 186.71 in GenBank (GenBank accession nos. KM199326 and KM199473), confirming the morphological identification. Phylogenetic reconstruction was generated by using the maximum likelihood (ML) method based on the Kimura 2-parameter model, with bootstrap nodal support for 1000 pseudoreplicates in MEGA software, version 7.0. The result showed that our isolates were clustered together with P. chamaeropis at 99% bootstrap values. Based on morphological characteristics and molecular phylogenetic analysis, the isolates were identified as P. chamaeropis. The pathogenicity of one representative isolate (JAUCC L001-1) was tested indoor by inoculating the top leaves of six healthy E. nitida plants. Three plants with three leaves were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and other three plants wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and non-infested PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box with high relative humidity at 25 °C. After 10 days, symptoms on all wounded inoculated plants were similar to those previously observed with distinct tiny circular spots, whereas no symptoms appeared on inoculated plants. Pestalotiopsis chamaeropis was re-isolated from symptomatic tissues but not from the mock-inoculated plants, and its identity was confirmed by morphological characteristics and molecular data, which confirmed Koch's postulates. Pestalotiopsis chamaeropis was previously reported as the causal agent of leaf blight diseases on Camellia sinensis in China (Chen et al. 2020), Pieris japonica in Japan (Nozawa et al. 2019) and Prostanthera rotundifolia in Australia (Azin et al. 2015). To our knowledge, this is the first report of P. chamaeropis causing a leaf spot disease on E. nitida in China, and this disease may be more widespread than the sampled location. This finds is beneficial to the better protection of E. nitida, a widespread medicinal and nectar source plant with high economic value.

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.

Genome-wide identification and characterization of long non-coding RNAs involved in flag leaf senescence of rice.

KEY MESSAGE: This study showed the systematic identification of long non-coding RNAs (lncRNAs) involving in flag leaf senescence of rice, providing the possible lncRNA-mRNA regulatory relationships and lncRNA-miRNA-mRNA ceRNA networks during leaf senescence. LncRNAs have been reported to play crucial roles in diverse biological processes. However, no systematic identification of lncRNAs associated with leaf senescence in plants has been studied. In this study, a genome-wide high throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. A total of 3953 lncRNAs and 38757 mRNAs were identified, of which 343 lncRNAs and 9412 mRNAs were differentially expressed. Through weighted gene co-expression network analysis (WGCNA), 22 continuously down-expressed lncRNAs targeting 812 co-expressed mRNAs and 48 continuously up-expressed lncRNAs targeting 1209 co-expressed mRNAs were considered to be significantly associated with flag leaf senescence. Gene Ontology results suggested that the senescence-associated lncRNAs targeted mRNAs involving in many biological processes, including transcription, hormone response, oxidation-reduction process and substance metabolism. Additionally, 43 senescence-associated lncRNAs were predicted to target 111 co-expressed transcription factors. Interestingly, 8 down-expressed lncRNAs and 29 up-expressed lncRNAs were found to separately target 12 and 20 well-studied senescence-associated genes (SAGs). Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 6 down-expressed lncRNAs possibly regulated 51 co-expressed mRNAs through 15 miRNAs, and 14 up-expressed lncRNAs possibly regulated 117 co-expressed mRNAs through 21 miRNAs. Importantly, by expression validation, a conserved miR164-NAC regulatory pathway was found to be possibly involved in leaf senescence, where lncRNA MSTRG.62092.1 may serve as a ceRNA binding with miR164a and miR164e to regulate three transcription factors. And two key lncRNAs MSTRG.31014.21 and MSTRG.31014.36 also could regulate the abscisic-acid biosynthetic gene BGIOSGA025169 (OsNCED4) and BGIOSGA016313 (NAC family) through osa-miR5809. The possible regulation networks of lncRNAs involving in leaf senescence were discussed, and several candidate lncRNAs were recommended for prior transgenic analysis. These findings will extend the understanding on the regulatory roles of lncRNAs in leaf senescence, and lay a foundation for functional research on candidate lncRNAs.

Systematic identification and characterization of circular RNAs involved in flag leaf senescence of rice.

MAIN CONCLUSION: Circular RNAs (circRNAs) identification, expression profiles, and construction of circRNA-parental gene relationships and circRNA-miRNA-mRNA ceRNA networks indicate that circRNAs are involved in flag leaf senescence of rice. Circular RNAs (circRNAs) are a class of 3'-5' head-to-tail covalently closed non-coding RNAs which have been proved to play important roles in various biological processes. However, no systematic identification of circRNAs associated with leaf senescence in rice has been studied. In this study, a genome-wide high-throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. Here, a total of 6612 circRNAs were identified, among which, 113 circRNAs were differentially expressed (DE) during the leaf senescence process. Moreover, 4601 (69.59%) circRNAs were derived from the exons or introns of their parental genes, while 2110 (71%) of the parental genes produced only one circRNA. The sequence alignment analysis showed that hundreds of rice circRNAs were conserved among different plant species. Gene Ontology (GO) enrichment analysis revealed that parental genes of DE circRNAs were enriched in many biological processes closely related to leaf senescence. Through weighted gene co-expression network analysis (WGCNA), six continuously down-expressed circRNAs, 18 continuously up-expressed circRNAs and 15 turn-point high-expressed circRNAs were considered to be highly associated with leaf senescence. Additionally, a total of 17 senescence-associated circRNAs were predicted to have parental genes, in which, regulations of three circRNAs to their parental genes were validated by qRT-PCR. The competing endogenous RNA (ceRNA) networks were also constructed. And a total of 11 senescence-associated circRNAs were predicted to act as miRNA sponges to regulate mRNAs, in which, regulation of two circRNAs to eight mRNAs was validated by qRT-PCR. It is discussed that senescence-associated circRNAs were involved in flag leaf senescence probably through mediating their parental genes and ceRNA networks, to participate in several well-studied senescence-associated processes, mainly including the processes of transcription, translation, and posttranslational modification (especially protein glycosylation), oxidation-reduction process, involvement of senescence-associated genes, hormone signaling pathway, proteolysis, and DNA damage repair. This study not only showed the systematic identification of circRNAs involved in leaf senescence of rice, but also laid a foundation for functional research on candidate circRNAs.

Analysis of Bacterial Communities in White Clover Seeds via High-Throughput Sequencing of 16S rRNA Gene.

White clover widely cultivated in China is one of the most important perennial leguminous forages in temperate and subtropical regions. There is a large quantity of white clover seeds imported into China each year for demands of high-quality grass seeds. Seedborne diseases may cause significant economic losses. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. Therefore, we used 16S rRNA gene sequencing to investigate the bacterial communities in white clover seeds collected from four different countries. The results showed that a total of 484,715 clean reads were obtained for further subsequent analysis. In total, 341, 340, 382, and 297 operational taxonomic units were obtained at 3% distance cutoff in DB, MB, TB, and XB samples, respectively. The richness indexes revealed that TB sample from Argentina had the highest bacterial richness in four samples. Our results demonstrated that Proteobacteria was the dominant phyla in MB, TB, and XB; however, Bacteroidetes was the dominant phyla in DB. The dominant genus of DB was Prevotella (11.9%), while Sphingomonas was the major genus of MB (46.9%), TB (55.08%), and XB (47.2%) samples. These results provide useful information for seedborne diseases and transmission of bacteria from seed to seedling.