First Report of Neofusicoccum parvum Causing Seedpod Blight on Lotus (Nelumbo nucifera) in China.

Lotus (Nelumbo nucifera Gaertn.) is a widely cultivated plant in China, and the fruit lotus variety has a high economic value attributed to the exquisite flavor of its fresh seeds. During the summer of 2023, an unidentified blight was observed affecting lotus seedpods in Jiande City, Zhejiang province, with approximately 65% of seedpods impacted in a 130-hectare area. The initial symptoms included dark purple spots on the lotus seedpod surface, which gradually expanded over time. After 5 to 7 days, the entire seedpod turned black, withering, and rendering the lotus seeds inedible. To identify the causal agent, tissues from symptomatic seedpods were excised and disinfected in 75% ethanol for 60 s, and washed twice in sterile distilled water. The disinfected symptomatic tissues (5 × 5 mm) were plated on potato dextrose agar (PDA), incubated at 25 ℃, transferred hyphal tips to obtain pure isolates after 3 days. Fungal colonies exhibiting Botryosphaeriaceae morphology were isolated from 33% of the samples (n = 15). Pure cultures were grown on PDA for both morphological and molecular identification. The colonies displayed a white aerial mycelium, turning olivaceous grey after 7 days. Pycnidia were produced within 3 weeks on PDA with added sterilized healthy lotus seedpod pieces on the surface. Conidia were hyaline, unicellular, ellipsoidal, 12.65 to 20.72 × 3.92 to 9.38 µm in size (mean 16.67 × 6.24 µm, n = 100). To determine the fungal species, genomic DNA was extracted from one representative isolate (ZJUP1112-1), to amplify four gene loci through polymerase chain reactions (PCR): rDNA internal transcribed spacer (ITS) with primers ITS1/ITS4, rDNA large subunit (LSU) with LR0R/LR5, the translation elongation factor 1-alpha gene (tef1) with EF1-728F/EF1-986R, and ß-tubulin gene (tub2) with Bt2a/Bt2b. The PCR products were Sanger sequenced in Zhejiang Shangya biotechnology co., LTD, and the resulting sequences were assembled and deposited in GenBank (ITS: OR740546; LSU: OR740547; tef1: OR776996; tub2: OR776997). BLAST searches indicated the highest nucleotide sequence identity with the reference strains of Neofusicoccum parvum CMW 9081 (ITS: 98.8%, AY236943; LSU: 100%, AY928045; tef1: 99.6%, AY236888; tub2: 99.3%, AY236917). Multi-locus phylogenetic analyses revealed that isolate ZJUP1112-1 formed a highly supported clade with N. parvum. Pathogenicity tests were performed on healthy lotus seedpods using mycelial plugs (5 mm diameter) from actively growing colonies of ZJUP1112-1 that were placed onto the front and side of the seedpods (6 each). Controls received PDA plugs. Treated seedpods were wrapped with parafilm and incubated at 25 ℃ and the experiment was repeated three times. After 5 days, dark purple lesions were observed on the inoculated seedpods, whereas controls remained symptomless. The same isolate was recovered from the margin of resulting lesions and confirmed by morphology, thus fulfilling Koch's postulates. N. parvum is a polyphagous pathogen causing blights and fruit rot on multiple economically important fruit crops, such as cacao (Puig et al. 2019), walnut (Chen et al. 2019), pistachio (Lopez-Moral et al. 2020), chestnut (Seddaiu et al. 2021), blueberry (Spetik et al. 2023) and mango (Polizzi et al. 2022), among others. To the best of our knowledge, this is the first report of N. parvum causing seedpod blight on lotus seedpods in China, which contributes to a better understanding of the pathogens affecting this plant species in China.

First Report of Monosporascus cannonballus causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China.

In June 2023, a sudden outbreak root rot and vine decline symptoms was observed during a watermelon (Citrullus lanatus T.) variety demonstration trial located in Taizhou City, Zhejiang Province, China, with an incidence rate ranging from 75% to 100% and an affected area of nearly 2,000 square meters. The disease initially appeared with a rapid and alarming invasion of root rot and vine decline symptoms within watermelon plants. Affected plants exhibited rapid deterioration, showing symptoms of wilting, yellowing and eventual demise, predominantly during the pre-harvest stage. Notably, numerous black, spherical, erumpent perithecia were clearly visible on the watermelon's root epidermis, a characteristic trait of the disease. Symptomatic plant samples were rigorously disinfected with 75% ethanol, and plated on potato dextrose agar medium for incubation at 25°C, successfully isolate two potential strains. These isolates were inoculated in oatmeal agar and incubated in a 25℃ light incubator. After 30 days, mature perithecia, the same as those found on the watermelon's root epidermis, reached a diameter of 500 µm. Each perithecium contained several pear-shaped asci, 56 to 108.5 µm in length and 30.5 to 46.4 µm in width, typically holding 1, rarely 2 ascospores. These characteristics align precisely with the typical strains of Monosporascus cannonballus Pollack and Uecker (1974). Additionally, sequencing the internal transcribed spacer region of ribosomal DNA (ITS) gene (White et al., 1990), large subunit ribosomal RNA (LSU) gene (Rehner and Samuels 1995), and beta-tubulin (TUB) gene (Glass and Donaldson, 1995) were performed. BLAST analysis indicated the highest nucleotide sequence identity with M. cannonballus CBS 586.93 reference sequence (ITS: 100%, JQ771930; TUB: 98.99%, JQ907292). Representative sequences of isolate ZJUP0990-2 from these regions were deposited in GenBank (Accession No.: OR357656 for ITS; OR474500 for LSU; OR365762 for TUB). A multigene phylogenomic analysis (ITS-LSU-TUB) was undertaken to ascertain the exact phylogenetic position of M. cannonballus within the genus Monosporascus. The amalgamation of both morphological and molecular insights consistently reaffirmed the accurate classification of the causative agent as M. cannonballus. To validate the pathogenicity of M. cannonballus, a controlled greenhouse experiment was conducted using watermelon (cv. Nabite) as the subject. Mycelium fragments, harvested from the edge of the colony ZJUP0990-2, were inoculated into oat liquid medium and cultivated under dark conditions at a consistent temperature of 30°C for 7 days. After 20 days, the inoculated plants exhibited root rot and wilting, mirroring the symptoms observed during the field outbreak. In contrast, the control plants did not exhibit any signs of disease. M. cannonballus was successfully re-isolated from the symptomatic roots of the inoculated plants, satisfying Koch's postulates. This experiment was repeated three times. This pathogenic fungus has previously been documented as a menace to melons in various regions including Mexico (Chew-Madinaveitia et al., 2012) and Brazil (Sales et al., 2004), as well as watermelons in Brazil (Sales et al., 2010), northern Mexico (Gaytan-Mascorro et al., 2012), and Saudi Arabia (Karlatti et al., 1997). To our knowledge, this is the first reported presence of M. cannonballus on watermelons in China. This new disease poses a serious threat to watermelon production, potentially leading to severe economic losses and impacting food security.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 µm x 3.77-6.84 µm (average 22.90 µm x 5.20 µm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.

Prevalence of Reversed Genome Organizations for Viruses in the Family Iflaviridae, Order Picornavirales.

Viruses in the order Picornavirales possess a positive-strand RNA genome that encodes structural proteins (SPs) and nonstructural proteins (NSPs). According to the recent report of the International Committee on Taxonomy of Viruses (ICTV), there are 8 families in Picornavirales, and monopartite picornaviruses in each family exhibit distinct types of genome organizations with rearranged genes coding for SPs and NSPs, namely, TypeI (5'-SPs-NSPs-3') and TypeII (5'-NSPs-SPs-3'). In the present study, 2 iflaviruses with the 2 genome types were unexpectedly identified in a damselfly host species, suggesting that these 2 genome types coexisted in the same host species, and the families of order Picornavirales might be more complex than previously thought. The consequent systematic homologous screening with all the publicly available picornaviruses successfully revealed a considerable number of candidates rearranged genome types of picornaviruses in various families of Picornavirales. Subsequently, phylogenetic trees were reconstructed based on RNA dependent RNA polymerase and coat protein, which evidently confirmed the prevalence of the 10 typeII iflaviruses in the Iflaviridae family. This suggests that genome types may not be relevant to viral taxonomy in this family. However, candidate picornaviruses with reversed genome types in the Secoviridae and Dicistroviridae families require further investigation. All in all, as the number of newly discovered viruses increases, more viruses with non-canonical genome arrangements will be uncovered, which can expand our current knowledge on the genome complexity and evolution of picornaviruses. IMPORTANCE Monopartite viruses in the order Picornavirales exhibit distinct genome arrangement of nonstructural proteins and structural proteins for each of the 8 families. Recent studies indicated that at least 4 ifla-like viruses possessed reversed genome organization in the family Iflaviridae, raising the possibility that this phenomenon may commonly present in different families of picornaviruses. Since we discovered 2 iflaviruses with exchanged structural and nonstructural proteins simultaneously in the damselfly, a systematic screening was subsequently performed for all of the current available picornaviruses (1,543 candidates). The results revealed 10 picornaviruses with reversed genome organization in the family Iflaviridae, implying that this phenomenon might prevalence in the order Picornavirales. These results will contribute to a better understanding for the future study on the genome complexity and taxonomy of picornaviruses.

Does the Relationship Between Bone Cement and the Intravertebral Cleft of Kummell Disease Affect the Efficacy of PKP?

OBJECTIVE: To study the relationship between distribution of bone cement and intravertebral cleft of patients with Kummell disease on the clinical effect of percutaneous kyphoplasty (PKP). METHODS: According to the relationship between the distribution of bone cement and the cleft in the vertebrae, a total of 92 patients with Kummell disease who underwent PKP in our hospital were divided into 2 groups. Specifically, the bone cement of patients in group A was localized in the cleft of the vertebrae and did not infiltrate around the cleft, while that of group B patients not only filled the cleft of the vertebrae, but also distributed diffusely around the cleft of the vertebrae. The amount of bone cement injected, leakage rate, visual analogue scale (VAS) score, Oswestry Disability Index (ODI), and vertebral imaging changes before operation, and 2 days and 1 year after operation were compared between the 2 groups. RESULTS: The amount of bone cement injected and the permeability of bone cement in group B were higher than those in group A (P < 0.05). The scores of VAS and ODI in both groups were significantly improved after operation, but the two scores in group B were better than those in group A one year after operation. The height of anterior vertebral body and Cobb's angle of kyphosis in the 2 groups were significantly improved after operation, but 1 year after operation, those in group B were better than those in group A. CONCLUSIONS: PKP was an effective method for treating Kummell disease. At the same time, the relationship between the distribution of bone cement and the cleft in the vertebral body was an important factor affecting the curative effect after PKP. The effect of the distribution pattern of bone cement filled with intravertebral cleft and diffusely distributed around the fissures was better than that of bone cement confined in the vertebral cleft.

MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Xuefu Zhuyu Oral Liquid () Prevents Apoptosis of Ischemic Myocardium Cells in Rats by Regulating SIRT1 and Its Pathway-Related Genes.

OBJECTIVE: To observe the changes of ischemic myocardial cells apoptosis in rats following intervention with Xuefu Zhuyu Oral Liquid (, XFZY), as well as changes of protein expression of silent information regulator 1 (SIRT1) and SIRT1 pathway-related genes. METHODS: H9c2 rat myocardial cells were divided into 6 groups: control group, oxygen glucose deprivation (OGD) group, SIRT1 siRNA group, OGD+SIRT1 siRNA group, OGD+XFZY group, and OGD+SIRT1 siRNA+XFZY group. Quantitative fluorescent polymerase chain reaction (PCR) and Western blot were used to detect the concentration variations of SIRT1 and its pathway-related genes and corresponding protein expression after XFZY intervention and SIRT1 transfection. RESULTS: Compared with the control group, the mRNA and protein expressions of SIRT1 were decreased obviously, while the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and nuclear factor kappa B (NF-ΚB) were increased in the OGD group, SIRT1 siRNA group, and OGD+SIRT1 siRNA group (P<0.01). Compared with the OGD group and OGD+SIRT1 siRNA group, the treatment of XFZY inhibited the decline in SIRT1 mRNA and protein expressions (P<0.01), and down-regulated the mRNA and protein levels of P53, FoxO1, FoxO3, FoxO4 and NF-ΚB, respectively (P<0.05 or P<0.01). CONCLUSION: XFZY could prevent myocardial cells apoptosis probably by increasing the mRNA and protein expressions of SIRT1 and inhibiting the mRNA and protein expressions of P53, NF- K B, FoxO1, FoxO3 and FoxO4.

MoFap7, a ribosome assembly factor, is required for fungal development and plant colonization of Magnaporthe oryzae.

Fap7, an important ribosome assembly factor, plays a vital role in pre-40S small ribosomal subunit synthesis in Saccharomyces cerevisiae via its ATPase activity. Currently, the biological functions of its homologs in filamentous fungi remain elusive. Here, MoFap7, a homologous protein of ScFap7, was identified in the rice blast fungus Magnaporthe oryzae, which is a devastating fungal pathogen in rice and threatens food security worldwide. ΔMofap7 mutants exhibited defects in growth and development, conidial morphology, appressorium formation and infection, and were sensitive to oxidative stress. In addition, site-directed mutagenesis analysis confirmed that the conserved Walker A motif and Walker B motif in MoFap7 are essential for the biological functions of M. oryzae. We further analyzed the regulation mechanism of MoFap7 in pathogenicity. MoFap7 was found to interact with MoMst50, a regulator functioning in the MAPK Pmk1 signaling pathway, that participates in modulating plant penetration and cell-to-cell invasion by regulating the phosphorylation of MoPmk1. Moreover, MoFap7 interacted with the GTPases MoCdc42 and MoRac1 to control growth and conidiogenesis. Taken together, the results of this study provide novel insights into MoFap7-mediated orchestration of the development and pathogenesis of filamentous fungi.

Identification and characterization of new Muscodor endophytes from gramineous plants in Xishuangbanna, China.

The endophytic fungi Muscodor spp. produce volatile organic compounds (VOCs) which can inhibit and even kill pathogenic fungi, bacteria, and nematodes. Nine endophytic fungal strains, isolated from the shoots of gramineous plants including Arthraxon hispidus, Eleusine indica, Oplismenus undulatifolius, and Oryza granulata, were identified as Muscodor through phylogenetic analysis of the internal transcribed spacer. Through an SPSS K-means cluster analysis, the nine Muscodor strains were divided into four groups based on the antifungal activities of the VOCs produced by these fungi determined by a two-section confrontation test. The first group contains the strains Y-L-54, W-S-41, Y-S-35, W-T-27, and Y-L-56, which showed the strongest activity. The second and third groups contain W-S-35 and Y-L-43, which showed stronger and moderate activity, respectively. The fourth group contains W-S-38 and N-L-7, which were the weakest in inhibiting the tested pathogens. Thirty-five compounds and the relative amounts of VOCs were determined by SPME-GC-MS and comparison with the NIST14 mass spectrometry database and Agilent MassHunter qualitative and quantitative analyses. These 35 compounds were classified into two different categories: (a) the product of fatty acid degradation, and (b) the intermediate and final metabolite of the metabolic pathway with the precursor of mevalonic acid. SPSS clustering analysis showed that the chemical components of VOCs might be correlated with their bioactivity rather than their phylogenetic assignment and some of the identified compounds might be responsible for antifungal activity. In conclusion, new Muscodor endophytes were recorded in tropical gramineous plants and a number of strains showed remarkable bioactive properties. Therefore, they have important potential applications in the fields of plant disease control.

Intravitreal anti-VEGF agents, oral glucocorticoids, and laser photocoagulation combination therapy for macular edema secondary to retinal vein occlusion: preliminary report.

AIM: To evaluate the efficacy and safety of combined anti-vascular endothelial growth factor (VEGF) agents, oral glucocorticoid, and laser photocoagulation therapy for macular edema (ME) secondary to retinal vein occlusion (RVO). METHODS: This study included 16 eyes of 16 patients with RVO-associated ME. Patients were initially treated with oral prednisone and an intravitreal anti-VEGF agent. Two weeks later, patients underwent standard laser photocoagulation. Best-corrected visual acuity (BCVA), central retinal thickness (CRT), and retinal vessel oxygenation were examined over 12mo. RESULTS: Patients received 1.43±0.81 anti-VEGF injections. Mean baseline and 12-month logMAR BCVA were 0.96±0.51 (20/178) and 0.31±0.88 (20/40), respectively, in eyes with central retinal vein occlusion (CRVO) (P<0.00), and 1.02±0.45 (20/209) and 0.60±0.49 (20/80), respectively, in eyes with branch retinal vein occlusion (BRVO) (P<0.00). At 12mo, CRT had significantly decreased in eyes with CRVO (P<0.00) and BRVO (P<0.00). Venous oxygen saturation had significantly increased in eyes with CRVO (P<0.00) and BRVO (P<0.00). No examined parameters were significantly different between the 2 RVO groups. No serious adverse effects occurred. CONCLUSION: Anti-VEGF, glucocorticoid, and photocoagulation combination therapy improves visual outcome, prolongs therapeutic effect, and reduces the number of intravitreal injections in eyes with RVO-associated ME.

[Pharmacological mechanism analysis of oligopeptide from Pinctada fucata based on in silico proteolysis and protein interaction network].

Thoracic obstruction is mainly attributed to the scope of coronary heart disease in modern medicine, and traditional Chinese medicine(TCM) shows a significant effect in the treatment of thoracic obstruction. In this research, a network pharmacology method was carried out to systemically study the underlying mechanism of the core herbal compatibility in TCM on the thoracic obstruction. First, we collected the literature about TCM prescriptions for treating thoracic obstruction from CNKI. Then, a prescription database was establish by TCM inheritance support platform system(V2.5) to determine the medication rules and core herbal compatibility in TCM. Finally, to obtain the potential signaling pathways, KEGG pathway analysis was performed by BATMAN-TCM online analysis tool. Results showed that the potential signal pathway of core herbal compatibility in TCM for the clinical treatment of thoracic obstruction was calcium ion and cGMP-PKG signaling pathway. This study provided a new research strategy for the study of the medication rules and mechanism of traditional Chinese medicine in the treatment of thoracic obstruction.

A Novel Derivative of (-)mycousnine Produced by the Endophytic Fungus Mycosphaerella nawae, Exhibits High and Selective Immunosuppressive Activity on T Cells.

An endophytic fungus, Mycosphaerella nawae ZJLQ129, was isolated from the leaves of the traditional Chinese medicine Smilax china. From the fermentation broth and mycelium, a dibenzofurane compound (-)mycousnine (1) was isolated. Chemical modification of it to the amide derivative (-)mycousnine enamine (2), which is new to science, was found to have high and selective immunosuppressive activity: similar to cyclosporin A, (-)mycousnine enamine (2) selectively inhibited T cell proliferation, suppressed the expression of the surface activation antigens CD25 and CD69 and the formation and expression of the cytokines interleukin-2 as well as interferon γ in activated T cells, but did not show any effect on the proliferation of B cells and cancer cells (PANC-1 and A549) and the activation of macrophages. Furthermore, the cytotoxicity of (-)mycousnine enamine was lower than that of cyclosporin A, and its therapeutic index (TC50/EC50) was 4,463.5, which is five-fold higher than that of cyclosporin A. We conclude that (-)mycousnine enamine (2), the semi-synthestic product prepared from the native product (-)mycousnine (1) of the endophyte M. nawae is a novel effective immunosuppressant showing low toxicity and high selectivity.

An autophagy gene, HoATG5, is involved in sporulation, cell wall integrity and infection of wounded barley leaves.

The endophytic fungus Harpophora oryzae is a beneficial endosymbiont isolated from wild rice. H. oryzae can not only promote rice growth and biomass accumulation but also protect rice roots from invasion by its close relative Magnaporthe oryzae. Autophagy is a highly evolutionary conserved process from lower to higher eukaryotic organisms, and is involved in the maintenance of normal cell differentiation and development. In this study, we isolated a gene (HoATG5) which encodes an essential protein required for autophagy from the beneficial endophyte fungus H. oryzae. Using targeted gene replacement, a ΔHoATG5 mutant was generated and used to investigate the biological functions of autophagy in H. oryzae. We found that the autophagic process was blocked in the HoATG5 deletion mutant. The mutant showed increased vegetative growth and sporulation, and was sensitive to nutrient starvation. The ΔHoATG5 mutant lost its ability to penetrate and infect the wounded barley leaves. These results provide new knowledge to elaborate the molecular machinery of autophagy in endophytic fungi.

Trichoderma Biodiversity of Agricultural Fields in East China Reveals a Gradient Distribution of Species.

We surveyed the Trichoderma (Hypocreales, Ascomycota) biodiversity in agricultural fields in four major agricultural provinces of East China. Trichoderma strains were identified based on molecular approaches and morphological characteristics. In three sampled seasons (spring, summer and autumn), 2078 strains were isolated and identified to 17 known species: T. harzianum (429 isolates), T. asperellum (425), T. hamatum (397), T. virens (340), T. koningiopsis (248), T. brevicompactum (73), T. atroviride (73), T. fertile (26), T. longibrachiatum (22), T. pleuroticola (16), T. erinaceum (16), T. oblongisporum (2), T. polysporum (2), T. spirale (2), T. capillare (2), T. velutinum (2), and T. saturnisporum (1). T. harzianum, T. asperellum, T. hamatum, and T. virens were identified as the dominant species with dominance (Y) values of 0.057, 0.052, 0.048, and 0.039, respectively. The species amount, isolate numbers and the dominant species of Trichoderma varied between provinces. Zhejiang Province has shown the highest diversity, which was reflected in the highest species amount (14) and the highest Shannon-Wiener diversity index of Trichoderma haplotypes (1.46). We observed that relative frequencies of T. hamatum and T. koningiopsis under rice soil were higher than those under wheat and maize soil, indicating the preference of Trichoderma to different crops. Remarkable seasonal variation was shown, with summer exhibiting the highest biodiversity of the studied seasons. These results show that Trichoderma biodiversity in agricultural fields varies by region, crop, and season. Zhejiang Province (the southernmost province in the investigated area) had more T. hamatum than Shandong Province (the northernmost province), not only in isolate amounts but also in haplotype amounts. Furthermore, at haplotype level, only T. hamatum showed a gradient distribution from south to north in correspondence analysis among the four dominant species. The above results would contribute to the application of Trichoderma biocontrol strains.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 µM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Friend or foe: differential responses of rice to invasion by mutualistic or pathogenic fungi revealed by RNAseq and metabolite profiling.

The rice endophyte Harpophora oryzae shares a common pathogenic ancestor with the rice blast fungus Magnaporthe oryzae. Direct comparison of the interactions between a single plant species and two closely-related (1) pathogenic and (2) mutualistic fungi species can improve our understanding of the evolution of the interactions between plants and fungi that lead to either mutualistic or pathogenic interactions. Differences in the metabolome and transcriptome of rice in response to challenge by H. or M. oryzae were investigated with GC-MS, RNA-seq, and qRT-PCR. Levels of metabolites of the shikimate and lignin biosynthesis pathways increased continuously in the M. oryzae-challenged rice roots (Mo-roots); these pathways were initially induced, but then suppressed, in the H. oryzae-challenged rice roots (Ho-roots). Compared to control samples, concentrations of sucrose and maltose were reduced in the Ho-roots and Mo-roots. The expression of most genes encoding enzymes involved in glycolysis and the TCA cycle were suppressed in the Ho-roots, but enhanced in the Mo-roots. The suppressed glycolysis in Ho-roots would result in the accumulation of glucose and fructose which was not detected in the Mo-roots. A novel co-evolution pattern of fungi-host interaction is proposed which highlights the importance of plant host in the evolution of fungal symbioses.

The rice endophyte Harpophora oryzae genome reveals evolution from a pathogen to a mutualistic endophyte.

The fungus Harpophora oryzae is a close relative of the pathogen Magnaporthe oryzae and a beneficial endosymbiont of wild rice. Here, we show that H. oryzae evolved from a pathogenic ancestor. The overall genomic structures of H. and M. oryzae were found to be similar. However, during interactions with rice, the expression of 11.7% of all genes showed opposing trends in the two fungi, suggesting differences in gene regulation. Moreover, infection patterns, triggering of host defense responses, signal transduction and nutritional preferences exhibited remarkable differentiation between the two fungi. In addition, the H. oryzae genome was found to contain thousands of loci of transposon-like elements, which led to the disruption of 929 genes. Our results indicate that the gain or loss of orphan genes, DNA duplications, gene family expansions and the frequent translocation of transposon-like elements have been important factors in the evolution of this endosymbiont from a pathogenic ancestor.

Evidence for biotrophic lifestyle and biocontrol potential of dark septate endophyte Harpophora oryzae to rice blast disease.

The mutualism pattern of the dark septate endophyte (DSE) Harpophora oryzae in rice roots and its biocontrol potential in rice blast disease caused by Magnaporthe oryzae were investigated. Fluorescent protein-expressing H. oryzae was used to monitor the colonization pattern. Hyphae invaded from the epidermis to the inner cortex, but not into the root stele. Fungal colonization increased with root tissue maturation, showing no colonization in the meristematic zone, slight colonization in the elongation zone, and heavy colonization in the differentiation zone. H. oryzae adopted a biotrophic lifestyle in roots accompanied by programmed cell death. Real-time PCR facilitated the accurate quantification of fungal growth and the respective plant response. The biocontrol potential of H. oryzae was visualized by inoculation with eGFP-tagged M. oryzae in rice. H. oryzae protected rice from M. oryzae root invasion by the accumulation of H2O2 and elevated antioxidative capacity. H. oryzae also induced systemic resistance against rice blast. This systemic resistance was mediated by the OsWRKY45-dependent salicylic acid (SA) signaling pathway, as indicated by the strongly upregulated expression of OsWRKY45. The colonization pattern of H. oryzae was consistent with the typical characteristics of DSEs. H. oryzae enhanced local resistance by reactive oxygen species (ROS) and high antioxidative level and induced OsWRKY45-dependent SA-mediated systemic resistance against rice blast.

An autophagy gene, MgATG5, is required for cell differentiation and pathogenesis in Magnaporthe oryzae.

Autophagy is a conserved degradation pathway that is involved in the maintenance of normal cell differentiation and development. The Saccharomyces cerevisiae ATG5 gene is an important component of the autophagy process. In this study, we identified MgATG5 as an autophagy-related gene in Magnaporthe oryzae that is homologous to ATG5. Using targeted gene replacement, an Mgatg5Delta mutant was generated and fungal autophagy was blocked. Cytological analysis revealed that the mutant had poor fungal morphogenic development, including a shortened aerial hyphae lifespan, decreased conidiation and perithecia formation, delayed conidial germination and appressorial formation, postponement of conidial cytoplasm transfer during appressorium formation, and reduction in formation of the penetration peg. Turnover of endogenous matter in the Mgatg5 mutant was also affected, as demonstrated by defects in the formation of conidial lipid droplets, and in the degradation of conidial glycogen deposits during appressorium formation. Lipid droplets and glycogen are necessary to generate adequate turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor and differentiation in the penetration peg, Mgatg5Delta pathogenicity was deficient in two host plants tested. The developmental and pathogenic phenotypes were restored by the introduction of an intact copy of MgATG5 into Mgatg5Delta, demonstrating that the MgATG5 deletion was responsible for the cellular defects. Taken together, these findings suggest that autophagy promotes cell differentiation through turnover of endogenous matter during fungal development, and is thus essential for the pathogenicity of the rice blast fungus.

Mnh6, a nonhistone protein, is required for fungal development and pathogenicity of Magnaporthe grisea.

Mnh6, a nonhistone protein containing an HMG1 box, was isolated from the rice blast fungus, Magnaporthe grisea. In the current study, we utilized an MNH6-deletion mutant to investigate the role of Mnh6 in the disease cycle of M. grisea. The Deltamnh6 mutant exhibited pleiotropic effects on fungal morphogenesis, including reduction in mycelial growth, conidiation, appressorium development, plant penetration, and infectious growth in host cells. Furthermore, Deltamnh6 mutant had greatly reduced pathogenicity on barley and rice compared to the wild-type. The reintroduction of an intact copy of MNH6 into the Deltamnh6 mutant restored morphological features and pathogenicity, suggesting that Mnh6 is required for fungal development, effective pathogenicity, and completion of the disease cycle of M. grisea.