Multiomics analysis reveals the molecular mechanisms underlying virulence in Rhizoctonia and jasmonic acid-mediated resistance in Tartary buckwheat (Fagopyrum tataricum).

Rhizoctonia solani is a devastating soil-borne pathogen that seriously threatens the cultivation of economically important crops. Multiple strains with a very broad host range have been identified, but only 1 (AG1-IA, which causes rice sheath blight disease) has been examined in detail. Here, we analyzed AG4-HGI 3 originally isolated from Tartary buckwheat (Fagopyrum tataricum), but with a host range comparable to AG1-IA. Genome comparison reveals abundant pathogenicity genes in this strain. We used multiomic approaches to improve the efficiency of screening for disease resistance genes. Transcriptomes of the plant-fungi interaction identified differentially expressed genes associated with virulence in Rhizoctonia and resistance in Tartary buckwheat. Integration with jasmonate-mediated transcriptome and metabolome changes revealed a negative regulator of jasmonate signaling, cytochrome P450 (FtCYP94C1), as increasing disease resistance probably via accumulation of resistance-related flavonoids. The integration of resistance data for 320 Tartary buckwheat accessions identified a gene homolog to aspartic proteinase (FtASP), with peak expression following R. solani inoculation. FtASP exhibits no proteinase activity but functions as an antibacterial peptide that slows fungal growth. This work reveals a potential mechanism behind pathogen virulence and host resistance, which should accelerate the molecular breeding of resistant varieties in economically essential crops.

Genome-Wide Identification of the WRKY Gene Family and Functional Characterization of CpWRKY5 in Cucurbita pepo.

The WRKY gene family is crucial for regulating plant growth and development. However, the WRKY gene is rarely studied in naked kernel formation in hull-less Cucurbita pepo L. (HLCP), a natural mutant that lacks the seed coat. In this research, 76 WRKY genes were identified through bioinformatics-based methods in C. pepo, and their phylogenetics, conserved motifs, synteny, collinearity, and temporal expression during seed coat development were analyzed. The results showed that 76 CpWRKYs were identified and categorized into three main groups (I-III), with Group II further divided into five subgroups (IIa-IIe). Moreover, 31 segmental duplication events were identified in 49 CpWRKY genes. A synteny analysis revealed that C. pepo shared more collinear regions with cucumber than with melon. Furthermore, quantitative RT-PCR (qRT-PCR) results indicated the differential expression of CpWRKYs across different varieties, with notable variations in seed coat development between HLCP and CP being attributed to differences in CpWRKY5 expression. To investigate this further, CpWRKY5-overexpression tobacco plants were generated, resulting in increased lignin content and an upregulation of related genes, as confirmed by qRT-PCR. This study offers valuable insights for future functional investigations of CpWRKY genes and presents novel information for understanding the regulation mechanism of lignin synthesis.

GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

Jasmonic acid-responsive RRTF1 transcription factor controls DTX18 gene expression in hydroxycinnamic acid amide secretion.

Jasmonates (JAs) are plant hormones that regulate the biosynthesis of many secondary metabolites, such as hydroxycinnamic acid amides (HCAAs), through jasmonic acid (JA)-responsive transcription factors (TFs). HCAAs are renowned for their role in plant defense against pathogens. The multidrug and toxic compound extrusion transporter DETOXIFICATION18 (DTX18) has been shown to mediate the extracellular accumulation of HCAAs p-coumaroylagmatine (CouAgm) at the plant surface for defense response. However, little is known about the regulatory mechanism of DTX18 gene expression by TFs. Yeast one-hybrid screening using the DTX18 promoter as bait isolated the key positive regulator redox-responsive TF 1 (RRTF1), which is a member of the AP2/ethylene-response factor family of proteins. RRTF1 is a JA-responsive factor that is required for the transcription of the DTX18 gene, and it thus promotes CouAgm secretion at the plant surface. As a result, overexpression of RRTF1 caused increased resistance against the fungus Botrytis cinerea, whereas rrtf1 mutant plants were more susceptible. Using yeast two-hybrid screening, we identified the BTB/POZ-MATH (BPM) protein BPM1 as an interacting partner of RRTF1. The BPM family of proteins acts as substrate adaptors of CUL3-based E3 ubiquitin ligases, and we found that only BPM1 and BPM3 were able to interact with RRTF1. In addition, we demonstrated that RRTF1 was subjected to degradation through the 26S proteasome pathway and that JA stabilized RRTF1. Knockout of BPM1 and BPM3 in bpm1/3 double mutants enhanced RRTF1 accumulation and DTX18 gene expression, thus increasing resistance to the fungus B. cinerea. Our results provide a better understanding of the fine-tuned regulation of JA-induced TFs in HCAA accumulation.

First report of brown rot of nectarine caused by Monilia yunnanensis in Tibet.

Brown rot caused by Monilinia spp. is one of the most important diseases of stone fruits. To date, three species of Monilinia have been found to occur on Prunus species worldwide: Monilinia fructicola (G. Winter) Honey, Monilinia fructigena (Aderhold & Ruhland) Honey, and Monilinia laxa (Aderhold & Ruhland) Honey (Zhu et al. 2005; Hu et al. 2011a). While M. fructicola is widespread in the Americas, and parts of Europe and Asia (CABI, 2010), M. laxa and M. fructigena are the primary species causing brown rot of peach in Europe (Bryde et al. 1977). In China, a new species Monilia yunnanensis was identified in 2011 (Hu et al. 2011b; Zhao et al. 2013; Yin et al. 2015; Yin et al. 2017). However, the species causing brown rot of nectarine (Prunus persica var. nectarina) in Tibet have not been undertaken. In the summer of 2017-2018, brown rot disease of nectarine was observed in Nyingchi, Tibet, and approximately 30% of nectarines were affected annually. Therefore, the brown rot disease of nectarine is one of the main factors that restrict the yield and quality of nectarine fruit production, and causes severe economic losses in Tibet. Thirty-six nectarine fruit with typical brown rot symptoms were collected from Tibet during the summer of 2017-2018. In order to isolate the causal agent, small pieces of pericarp were disinfected with 75% ethanol for 1 min, and then for 1 min in 1% NaOCl, rinsed in sterile distilled water for three times, dried on sterile paper and placed on potato dextrose agar (PDA). Thirty-six single-spore isolates were obtained and all morphologically similar, and three representative isolates 2-1, 2-16 and 2-31 which were from different period and years in 2017-2018 were characterized phylogenetically and morphologically to identify them to species level. Pathogenicity of each representative isolate was confirmed by inoculating five surface-disinfected mature nectarines with mycelial plugs in the wound of the fruit. Nectarine fruit inoculated with sterile PDA plugs served as the negative control. The inoculated nectarines developed brown lesions after 6 days incubation at 22°C, and the pathogen was successfully re-isolated. There were no symptoms on the control nectarine fruit. The isolates 2-1, 2-16 and 2-31 produced gray-green colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the media after 16 days of incubation at 22°C, resembling those described for M. yunnanensis (Hu et al. 2011b). Conidia were one-celled, hyaline, ellipsoid to lemon shape (9.24 to 15.58 µm), and borne in branched monilioid chains. The average daily growth of mycelium on PDA at 22°C was 11.56 mm. Therefore, the isolates 2-1, 2-16 and 2-31 were preliminarily identified as M. yunnanensis based on the morphological investigations (Hu et al. 2011b). Morphological identification was confirmed by phylogenetic analysis based on sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and ß-tubulin (TUB2) genes of 2-1, 2-16 and 2-31 which were amplified using primers Mon-G3pdhF/Mon-G3pdhR and Mon-TubF1/Mon-TubR1 (Hu et al. 2011b). In both G3PDH and TUB2 phylogenetic trees, the isolates 2-1, 2-16 and 2-31 formed monophyletic clades within a derived clade with the M. yunnanensis isolates. Additionally, the three isolates were more closely related to M. yunnanensis (HQ908782.1 and HQ908783.1) than to other Monilinia species. Based on morphological and molecular identification, the isolates 2-1, 2-16 and 2-31 were identified as M. yunnanensis. Previously, M. yunnanensis has been reported as a new species causing brown rot of peach in China (Hu et al, 2011b). To our knowledge, this is the first report of M. yunnanensis causing nectarine fruit brown rot in Tibet. These findings suggest that M. yunnanensis is spreading on its principal host plants and causing substantial economic losses in the Tibet fruit production.

First report of Rhizoctonia solani AG-4 HGI Causing Stem Canker on Fagopyrum tataricum (Tartary buckwheat) in China.

Tartary buckwheat (Fagopyrum tataricum) is an ideal functional food source, which is well known to be gluten-free and rich in proteins, fats, vitamins, minerals, and flavonoids of various pharmaceutical uses, such as rutin, quercitin and epicatechin (Zhou, M et al. 2018). The Rhizoctonia solani AG-4 HGIII causing severe canker disease was first isolated from common buckwheat (F. esculentum) in Inner Mongolia of China (Zhou, H et al. 2015). In 2018, sunken lesion and dark brown symptoms were observed on the root and stem of ten days old Tartary buckwheat in Liangshan (28°21'N, 103°19''E), Sichuan Province and Fenghuang (28°19' N, 109°48' E), Hunan Province in China. In the beginning, water soaked spots appeared on the stem base, where gradually became rotten and necrotic, finally resulting in the damping-off and death of buckwheat seedlings. This disease had over 40% incidence and lead to serious losses to the buckwheat production in 2018. To isolate the pathogens on Tartary buckwheat, ten plants with typical symptoms were collected from each location. The infected tissue was taken and cut into 3mm pieces from the margin between healthy and diseased tissue, surface sterilized with 1% sodium hypochlorite solution for 4 min, washed three times with sterile distilled water, dried on sterilized filter paper and then placed on potato dextrose agar (PDA) with 100 mg/ml streptomycin sulfate. After incubation at 28℃ in the dark for 2 days, mycelial tips of four fungal cultures were transferred to PDA plates for purification. Initially, colonies were pale white, and then turned brown after 2 days incubation. The mycelium was hairy and concentrically whorled in the culture medium. Microscopic observation showed that the hyphae characteristically branched at right angles and had constriction at the base of hyphal branches. Nuclear staining showed that the hyphae cells were multinuclear. These morphological features revealed that the isolates belonged to R. solani (Sneh et al. 1991). Subsequently, the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of one isolate was amplified by PCR (White et al. 1990) and sequenced (GeneBank accession no. MT078642) by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. DNA was extracted by Fungal genomic DNA Extraction Kit, D3390-02, OMEGA. The BLAST similarity analysis showed a 99.96% match with R. solani AG-4 HGI (GenBank accession no. JQ343830) and 99.85% identity to R. solani AG-4 HGI isolate SX-8 (GenBank accession no. KJ170346) (Suli, Sun et al. 2015). Furthermore, the phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the isolate was clearly clustered with the group of R. solani AG-4 HGI (Ireland et al. 2015). Pathogenicity was tested in the greenhouse condition to satisfy Koch's postulates. Tartary buckwheat plants of seven days old and fifty days old were respectively inoculated near the base of the stem neck with one mycelial plug contacted directly. Ten plants in a pot were inoculated as one treatment, four pots were used for replicates. Control plants were inoculated with PDA medium plugs without fungi. All the plants were kept at 26℃ with 14 h light, 10 h dark and 96% humidity. After five days (Suli, Sun et al. 2015), over 90% of the inoculated plants exhibited necrotic brown lesions on stems that was similar to those symptoms observed in the field, whereas control plants remained asymptomatic. The visible characteristics and ITS sequence of the pathogen re-isolated from symptomatic plants were in accordance with the original isolate (R. solani AG-4 HGI). Based on disease symptoms in the fields, morphological characteristics, ITS sequence analysis, and pathogenicity assay, we concluded that R. solani AG-4 HGI was the principle cause of Tartary buckwheat blight in Liangshan, Sichuan Province and Fenghuang, Hunan Province in China. Previously, R. solani AG-4 HGI has been identified as a Chinese chive pathogen (Shi, Y et al. 2017). To the best of our knowledge, this is the first report of the natural occurrence of Rhizoctonia solani AG-4 HGI affecting Tartary Buckwheat in China. This finding is helpful for the early diagnosis and identification of the disease, which will be the guiding of effective control methods to the devastating disease at the early stage.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Hulless Cucurbita pepo of Inner Mongolia and Xinjiang Autonomous Region in China.

Hulless Cucurbita pepo is an annual herb in the Cucurbitaceae family and is one of the main economic vegetable crops in China, and is a raw material for cosmetics and health care products. It is also called hulless pumpkin because its seeds have no seed coat, which is a rare variation of the Cucurbita. In July 2010, powdery mildew was observed on hulless Cucurbita pepo 'Tianran' in fields of Wuwei District, China(Liang et al.2010). Disease incidence when first observed was 65.67%, but increased to 100% in July 2019. Early disease symptoms appeared as circular or irregular white powdery areas on both leaf surfaces. At later infection stages, entire leaves，petioles, and stems were covered with white fungal mycelia that resulted in leaf yellowing and senescence，but not defoliation . Fungal hyphae were septate, branched and flexuous to straigh. Conidiophores were unbranched, straight and grew vertically to the mycelium. Conidiophore foot cells of the were cylindrical with slight constriction at basal septa and followed by one to four short cells that eventually became conidia. Conidia were barrel-shaped with ends darker than the middle and measured 20 to 32 × 12 to 19µm.T Cleistothecium formed at 20 ℃, 70% relative humidity and light intensity of 4,400 lx. They were scattered, spherical, dark brown, with parietal cells irregularly rectangular or polygonal, and 70 to 75×90 to 95µm in diameter. Cleistothecia had four to eight appendages that were colorless to partially brown, each with three to five septa and lengths 0.5 to 3.0 times the diameter of cleistothecia. The powdery mildew fungus was tentatively identified as Podosphaera xanthii and showed characteristics similar to those reported by others (Cui et al. 2018; Choi et al. 2020). rDNA was extracted from pools of fungus conidia and the ITS region amplified using primers ITS 1:5'-TCCGTAGGT GAACCTGCGG-3'/ITS 4:5'-TCCTCCGCTTATTGATATGC-3' and then sequenced. BLASTn analysis of the 540-bp (MT250855) amplicon revealed 100% sequence identity with respective rDNA sequences of Podosphaera xanthii isolates from Momordica charantia(AB774158.1). Based on the morphological characteristics and ITS sequences, the fungal species was identified as P. xanthii .Pathogenicity of the powdery mildew fungus was tested by dusting conidia from infected hulless pumpkin leaves onto three asymptomatic plants. Three noninoculated plants was used as a controls. The Infection process of P.xanthii on pumpkins observed that the conidia began to germinate at 12h after inoculation.Twenty four to 72 h post inoculation, powdery mildew mycelia appeared on inoculated leaves. After 73 to 96 h, chains of conidia formed and these germinated to form secondary infection sites. In later stages of the disease cycle, dark brown cleistothecia formed on the yellowing plant foliage. Fungus morphology from inoculated leaves was identical to that observed on original naturally infected plants. Uninoculated plants remained healthy. Powdery mildew caused by P. xanthii is a major foliage disease that affects members of the Cucurbitaceae family worldwide. The fungus has previously been reported from China on Cucurbita moschata (DQ490752), Cucurbita maxima (DQ490759), Cucurbita pepo(DQ490750), Cucumis sativus(DQ490755)(Park et al.2010; Liang et al.2007) and Cucurbita maxima, Abelmoschus esculentus (okra), Sechium edule (mirliton, vegetable pear), and Lagenaria siceraria (bottle gourd) (Choi et al. 2020; Fan et al. 2019; Xu et al. 2020; Cui et al. 2018).To our knowledge, this is the first report on the occurrence of P. xanthii on hulless Cucurbita pepo in China.

Mechanisms of the IAA and ACC-deaminase producing strain of Trichoderma longibrachiatum T6 in enhancing wheat seedling tolerance to NaCl stress.

BACKGROUND: Trichoderma species, a class of plant beneficial fungi, may provide opportunistic symbionts to induce plant tolerance to abiotic stresses. Here, we determined the possible mechanisms responsible for the indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate-deaminase (ACC-deaminase) producing strain of Trichoderma longibrachiatum T6 (TL-6) in promoting wheat (Triticum aestivum L.) growth and enhancing plant tolerance to NaCl stress. RESULTS: Wheat treated with or without TL-6 was grown under different levels of salt stress in controlled environmental conditions. TL-6 showed a high level of tolerance to 10 mg ml- 1 of NaCl stress and the inhibitory effect was more pronounced at higher NaCl concentrations. Under NaCl stress, the activity of ACC-deaminase and IAA concentration in TL-6 were promoted, with the activity of ACC-deaminase increased by 26% at the salt concentration of 10 mg ml- 1 and 31% at 20 mg ml- 1, compared with non-saline stress; and the concentration of IAA was increased by 10 and 7%, respectively (P < 0.05). The increased ACC-deaminase and IAA concentration in the TL-6 strain may serve as an important signal to alleviate the negative effect of NaCl stress on wheat growth. As such, wheat seedlings with the ACC-deaminase and IAA producing strain of TL-6 treatment under NaCl stress increased the IAA concentration by an average of 11%, decreased the activity of ACC oxidase (ACO) by an average of 12% and ACC synthase (ACS) 13%, and decreased the level of ethylene synthesis and the content of ACC by 12 and 22%, respectively (P < 0.05). The TL-6 treatment decreased the transcriptional level of ethylene synthesis genes expression, and increased the IAA production genes expression significantly in wheat seedlings roots; down-regulated the expression of ACO genes by an average of 9% and ACS genes 12%, whereas up-regulated the expression of IAA genes by 10% (P < 0.05). TL-6 treatments under NaCl stress decreased the level of Na+ accumulation; and increased the uptake of K+ and the ratio of K+/Na+, and the transcriptional level of Na+/H+ antiporter gene expression in both shoots and roots. CONCLUSIONS: Our results indicate that the strain of TL-6 effectively promoted wheat growth and enhanced plant tolerance to NaCl stress through the increased ACC-deaminase activity and IAA production in TL-6 stain that modulate the IAA and ethylene synthesis, and regulate the transcriptional levels of IAA and ethylene synthesis genes expression in wheat seedling roots under salt stress, and minimize ionic toxicity by disturbing the intracellular ionic homeostasis in the plant cells. These biochemical, physiological and molecular responses helped promote the wheat seedling growth and enhanced plant tolerance to salt stress.

Seed Treatment with Trichoderma longibrachiatum T6 Promotes Wheat Seedling Growth under NaCl Stress Through Activating the Enzymatic and Nonenzymatic Antioxidant Defense Systems.

Salt stress is one of the major abiotic stresses limiting crop growth and productivity worldwide. Species of Trichoderma are widely recognized for their bio-control abilities, but little information is regarding to the ability and mechanisms of their promoting plant growth and enhancing plant tolerance to different levels of salt stress. Hence, we determined (i) the role of Trichoderma longibrachiatum T6 (TL-6) in promoting wheat (Triticum aestivum L.) seed germination and seedling growth under different levels of salt stress, and (ii) the mechanisms responsible for the enhanced tolerance of wheat to salt stress by TL-6. Wheat seeds treated with or without TL-6 were grown under different levels of salt stress in controlled environmental conditions. As such, the TL-6 treatments promoted seed germination and increased the shoot and root weights of wheat seedlings under both non-stress and salt-stress conditions. Wheat seedlings with TL-6 treatments under different levels of NaCl stress increased proline content by an average of 11%, ascorbate 15%, and glutathione 28%; and decreased the contents of malondialdehyde (MDA) by an average of 19% and hydrogen peroxide (H2O2) 13%. The TL-6 treatments induced the transcriptional level of reactive oxygen species (ROS) scavenging enzymes, leading to the increases of glutathione s-transferase (GST) by an average of 17%, glutathione peroxidase (GPX) 16%, ascorbate peroxidase (APX) 17%, glutathione reductase (GR) 18%, dehydroascorbate reductase (DHAR) 5%. Our results indicate that the beneficial strain of TL-6 effectively scavenged ROS under NaCl stress through modulating the activity of ROS scavenging enzymes, regulating the transcriptional levels of ROS scavenging enzyme gene expression, and enhancing the nonenzymatic antioxidants in wheat seedling in response to salt stress. Our present study provides a new insight into the mechanisms of TL-6 can activate the enzymatic and nonenzymatic antioxidant defense systems and enhance wheat seedling tolerance to different levels of salt stress at physiological, biochemical and molecular levels.

Sphingobacterium solani sp. nov., isolated from potato stems.

A Gram-stain-negative, non-motile, non-spore-forming bacterium, designated MLS-26-JM13-11T, was isolated from potato stems, collected in Guyuan County, Hebei Province, China. Strain MLS-26-JM13-11T could grow at 10-39 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.2) and in the presence of 0-4.0 % (w/v) NaCl (optimum, 1.0 % w/v). Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain MLS-26-JM13-11T formed a stable clade with Sphingobacterium bambusae IBFC2009T and Sphingobacterium griseoflavum SCU-B140T, with the 16S rRNA gene sequence similarities ranging from 95.9 % to 97.0 %. The major cellular fatty acids comprised iso-C15 : 0 (36.9 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c, 34.0 %), C16 : 0 (3.0 %) and iso-C17 : 0 3-OH (13.4 %). Strain MLS-26-JM13-11T contained sphingoglycolipid, phosphatidyl ethanolamine, six unknown lipids, one unknown aminolipid, four unknown polarlipids and two unknown aminophospholipids. The isoprenoid quinone was MK-7. The DNA G+C content was 42.6 mol%. Furthermore, the average nucleotide identity and in silico estimated DNA-DNA reassociation values among MLS-26-JM13-11T and S. bambusae KCTC 22814T were in all cases below the respective threshold for species differentiation. On the basis of phenotypic, genotypic and phylogenetic evidence, strain MLS-26-JM13-11T (=ACCC 60057T=JCM 32274T) represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium solani sp. nov. is proposed.

Identification of the antifungal activity of Trichoderma longibrachiatum T6 and assessment of bioactive substances in controlling phytopathgens.

Biological control with microbial antagonists is considered an alternative approach for controlling plant diseases. Trichoderma species are one of the potential fungal biocontrol agents in suppression of soil-borne pathogens. However, the mechanism and characterization of Trichoderma spp. in inhibiting different phytopathogenic fungi are largely unknown. In this study, we investigated the antagonistic potential of the endophytic fungus Trichoderma longibrachiatum T6 as a biocontrol agent against different phytopathogenic fungi and the associated antagonistic mechanism with bioactive substances. We found that the fermentation and crude extract of T. longibrachiatum T6 had a broad spectrum and potent activity inhibiting the growth of eleven phytopathogens evaluated, and of which, the inhibitory rate against Valsa mali reached 95% at 5 days after incubation. Ten fractions and six sub-fractions of bioactive substances were obtained on silica gel G chromatography and Sephadex LH-20 columns. One of the sub-fractions (coded sub-Fr.4f) exhibited highest inhibition against the pathogen V. mail, with the inhibitory rate of 80.64% at Day 5 of the treatment. Four key chemical inhibitors were identified: (i) 1, 2-Benzenedicarboxylicacid, bis (2-methylpropyl) ester (DIBP) (C16H22O4); (ii) (Z)-octadec-9-enoic acid (C18H34O2); (iii) 1, 2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (MEHP) (C16H22O4); and (iv) (Z)-13-Docosenamide (C22H43NO), using spectroscopic and nuclear magnetic resonance data. Two fungicidal compounds DIBP and MEHP provided significantly greater antifungal activities than the other compounds in the inhibition of the V. mail growth. There was a significant linear relationship between the monomer compounds MEPH or DIBP and the inhibitory rates of V. mail; at the concentration of 200 µg mL-1, the inhibitory rate reached over 86% or 78%. We conclude that the strain of T. longibrachiatum T6 can serve as an effective biocontrol agent against V. mali and the mechanism for this function was due to the secondary metabolites with effective bioactive substance.

[Parasitic and lethal action of Trichoderma longibrachiatum against Heterodera avenae].

OBJECTIVE: To evaluate the potential of Trichoderma longibrachiatum spore suspension against Heterodera avenae. METHODS: The parasitic and lethal effects of T. longibrachiatum spore suspension against the cysts of H. avenae were studied in vitro and observed under microscope. RESULTS: Microscopic observation showed that the spore suspension of T. longibrachiatum parasitized on the cyst surface, germinated a large number of hyphae, and grew on the surface of the cyst at the initial stage. Later, the cysts were completely surrounded by dense mycelium, and the contents of digestion in cysts was lysed, even some cysts produced vacuoles, and some were split up and finally the cyst was dissolved by the metabolite of T. longibrachiatum. In vitro studies showed that high concentrations of T. longibrachiatum spores had strong parasitic and lethal effects on the cysts of H. avenae, and the probable mechanism of parasitic and lethal effects of T. longibrachiatum against H. avenae were mainly by inducing and increasing chitinase, glucanase and caseinase activity. The cysts were parasitized by 93.3% at 18 days, the hatching of cysts were inhibited by 93.6% at 10 days when treated with the concentrations (1.5 x 10(8) CFU/mL) of T. longibrachiatum. CONCLUSION: Trichoderma longibrachiatum had strong parasitic and lethal effects on the cysts of H. avenae, and has the potential as a new biocontrol agent.

Characterization of the Serine Protease TlSP1 from Trichoderma longibrachiatum T6 and Its Function in the Control of Heterodera avenae in Wheat.

Serine protease is an extracellular protease secreted by biocontrol fungi that can effectively control nematode diseases by degrading nematode eggshells and enhancing plant resistance. Trichoderma longibrachiatum T6, an important biocontrol fungus, has been demonstrated to effectively parasitize and degrade Heterodera avenae cysts, eggs, and second-stage juveniles (J2s). However, the genes that encoding serine protease and their functions in T. longibrachiatum T6 have not been thoroughly investigated. In this study, we successfully cloned and sequenced the serine protease gene TlSP1 in T. longibrachiatum T6. Our results revealed that the expression level of the TlSP1 gene was induced and significantly increased in T. longibrachiatum T6 after inoculation with H. avenae cysts. The full-length sequence of the coding region (CDS) of TlSP1 gene was 1230 bp and encoded a protein consisting of 409 amino acids. Upon the transformation of the TlSP1 gene into Pichia pastoris X33, the purified recombinant TlSP1 protein exhibited optimal activity at a temperature of 50 °C and pH 8.0. Following 4-10-day of treatment with the purified recombinant TlSP1 protein, the eggshells and content were dissolved and exuded. The number of nematodes invading wheat roots was reduced by 38.43% in the group treated with both TlSP1 and eggs on one side (P1+N) compared to the control group, while the number of nematodes invading wheat roots was reduced by 30.4% in the TlSP1 and eggs two-sided treatment group (P1/N). Furthermore, both the P1+N and P1/N treatments significantly upregulated genes associated with defense enzymes (TaPAL, TaCAT, TaSOD, and TaPOD), genes involved in the lignin synthesis pathway (TaC4H, Ta4CL2, TaCAD1, and TaCAD12), and salicylic acid (SA)-responsive genes (TaNPR1, TaPR1, and TaPR2) and led to the high expression of jasmonic acid (JA)-responsive genes (TaPR4, TaOPR3, and TaAOS2). This study has highlighted the significant role of the TlSP1 gene in facilitating H. avenae eggshells' dissolution, preventing nematode invasion in the host plant, and boosting plant resistance in wheat.

Identification the Pathogen Cause a New Apple Leaf Blight in China and Determination the Controlling Efficacy for Five Botanical Fungicides.

Alternaria leaf blight has recently been described as an emerging fungal disease of apple trees which is causing the significant damage in the apple-growing areas of Tianshui and Jingning, Gansu, China. In the present study, the pathogen species involved in apple leaf blight and its biological characteristics were identified, and the inhibitory activity of different botanical fungicides against the pathogen was evaluated in vitro. Four strains were isolated from the symptomatic areas of necrotic apple leaves, and initially healthy leaves showed similar symptoms to those observed in orchards after inoculation with the ABL2 isolate. The ABL2 isolate was identified as Alternaria tenuissima based on the morphological characteristics of its colonies, conidiophores, and conidia, and this was also confirmed by multi-gene sequence (ITS, OPA10-2, Alta-1, and endoPG) analysis and phylogenic analysis. The optimum temperature, pH, carbon source, and nitrogen source for the growth of A. tenuissima mycelia were 28 °C, 6-7, soluble starch, and soy flour, respectively. In addition, the botanical fungicide eugenol exhibited the highest inhibitory effect on the mycelial growth and conidia germination of A. tenuissima, and the median effective concentration (EC50) values were 0.826 and 0.755 µg/mL, respectively. The protective and curative efficacy of eugenol were 86.85% and 76.94% after inoculation in detached apple leaves at a concentration of 4 µg/mL. Our research provides new insights into the control of apple leaf blight disease by applying botanical fungicides.

Characterization and Pathogenicity of Colletotrichum truncatum Causing Hylocereus undatus Anthracnose through the Changes of Cell Wall-Degrading Enzymes and Components in Fruits.

Anthracnose is one of the destructive diseases of pitaya that seriously affects the plant growth and fruit quality and causes significant yield and economic losses worldwide. However, information regarding the species of pathogens that cause anthracnose in pitaya (Hylocereus undatus) fruits in Gansu Province, China, and its pathogenic mechanism is unknown. Thus, the purposes of our present study were to identify the species of pathogens causing H. undatus fruits anthracnose based on the morphological and molecular characteristics and determine its pathogenic mechanism by physiological and biochemical methods. In our present study, forty-six isolates were isolated from the collected samples of diseased H. undatus fruits and classified as three types (named as H-1, H-2, and H-3), according to the colony and conidium morphological characteristics. The isolation frequencies of H-1, H-2, and H-3 types were 63.04%, 21.74%, and 15.22%, respectively. The representative single-spore isolate of HLGTJ-1 in H-1 type has significant pathogenicity, and finally we identified Colletotrichum truncatum as the pathogen based on the morphological characteristics as well as multi-locus sequence analysis. Moreover, the H. undatus fruits inoculated with C. truncatum had a significantly increased activity of cell wall-degrading enzymes (CWDEs) cellulase (Cx), ß-glucosidase (ß-Glu), polygalacturonase (PG), and pectin methylgalacturonase (PMG), while having a decreased level of cell wall components of original pectin and cellulose in comparison to control. The average increased activities of Cx, ß-Glu, PG, and PMG were 30.73%, 40.40%, 51.55%, and 32.23% from day 0 to 6 after inoculation, respectively. In contrast, the average decreased contents of original pectin and cellulose were 1.82% and 16.47%, respectively, whereas the average increased soluble pectin content was 38.31% in comparison to control. Our results indicate that C. truncatum infection increased the activities of CWDEs in H. undatus fruits to disassemble their cell wall components, finally leading to the fruits' decay and deterioration. Thus, our findings will provide significant evidence in the controlling of pitaya anthracnose in the future.

Deep Multi-Modal Discriminative and Interpretability Network for Alzheimer's Disease Diagnosis.

Multi-modal fusion has become an important data analysis technology in Alzheimer's disease (AD) diagnosis, which is committed to effectively extract and utilize complementary information among different modalities. However, most of the existing fusion methods focus on pursuing common feature representation by transformation, and ignore discriminative structural information among samples. In addition, most fusion methods use high-order feature extraction, such as deep neural network, by which it is difficult to identify biomarkers. In this paper, we propose a novel method named deep multi-modal discriminative and interpretability network (DMDIN), which aligns samples in a discriminative common space and provides a new approach to identify significant brain regions (ROIs) in AD diagnosis. Specifically, we reconstruct each modality with a hierarchical representation through multilayer perceptron (MLP), and take advantage of the shared self-expression coefficients constrained by diagonal blocks to embed the structural information of inter-class and the intra-class. Further, the generalized canonical correlation analysis (GCCA) is adopted as a correlation constraint to generate a discriminative common space, in which samples of the same category gather while samples of different categories stay away. Finally, in order to enhance the interpretability of the deep learning model, we utilize knowledge distillation to reproduce coordinated representations and capture influence of brain regions in AD classification. Experiments show that the proposed method performs better than several state-of-the-art methods in AD diagnosis.

Trichoderma longibrachiatum T6: A nematocidal activity of endochitinase gene exploration and its function identification.

Endochitinase is a natural extracellular protein in Trichoderma longibrachiatum T6, which can degrade the eggshell of Heterodera avenae significantly, however the related genes that coding this protein was rarely characterized. In the present study, the endochitinase 18-5 gene (T6-Echi18-5) of T. longibrachiatum T6 was cloned and sequenced. The expression level of T6-Echi18-5 gene in T. longibrachiatum T6 was induced and increased after the H. avenae cysts inoculation. The full-length cDNA sequence of T6-Echi18-5 was 1671 bp that contained an ORF of 1275 bp, corresponding to 424 amino acids with a 45.9 kDa molecular weight. A single band of 60.04 kDa was detected and identified using SDS-PAGE and Western blot analysis after transferring the T6-Echi18-5 gene to Escherichia coli BL21 Rosetta (DE3). The concentration of purified recombinant T6-Echi18-5 protein was 1.53 mg·ml-1, and the optimal temperature and pH were 50 °C and 5.0, respectively. The eggshell and content were dissolved and exuded from 4 to10 days after treatment with the purified recombinant T6-Echi18-5 protein. The relative inhibition rate of eggs hatching was 86.79 % at 12 days after treatment. Our study demonstrated the key role of T6-Echi18-5 gene in degrading the H. avenae eggshell and inhibiting the eggs hatching.

Mortality, Enzymatic Antioxidant Activity and Gene Expression of Cabbage Aphid (Brevicoryne brassicae L.) in Response to Trichoderma longibrachiatum T6.

Aphids are one of the most common insect pests in greenhouse and field crops worldwide, causing significant crop yields and economic losses. The objective of this study was to determine the mortality, enzymatic antioxidant activity and gene expression of cabbage aphids (Brevicoryne brassicae L.) in response to Trichoderma longibrachiatum T6 (T6) at different time points from Day 1 to 7 after inoculation. Our results showed that the highest mortality of B. brassicae was observed on Day 7 at a concentration of 1 × 108 spores ml-1 (73.31%) after inoculation with T6 compared with the control on Day 7 (11.51%). The activities of the enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione S-transferase (GST) were increased by 52.98%, 44.77%, 48.26%, 49.39%, 45.13% and 39.67%, respectively on Day 3 after inoculation with T6 compared to the control. Howerver increased days post treatment (dpt) decreased the activities of SOD, POD, CAT, APX, GPX and GST enzymes by 20.79%, 21.05%, 13.75%, 20.45%, 25.38%, and 19.76% repectively on Day 7 compared to control. The transcript levels of SOD, POD, CAT, GPX, and GST genes were increased by 10.87, 9.87, 12.77, 6.22 and 4.07 respectively at Day 3 after inoculation with T6 in comparison to the control. However, the SOD, POD, CAT, GPX, and GST transcription levels decreased by 0.43, 0.44, 0.35, 0.52 and 0.47 respectively, compared to control at Day 7. Our results suggest that the T6 strain has a potential effect on the antioxidant activity and mortality of B. brassicae and therefore could be used as a natural biocontrol agent against B. brassicae in the future.

Mechanisms of Trichoderma longibrachiatum T6 Fermentation against Valsa mali through Inhibiting Its Growth and Reproduction, Pathogenicity and Gene Expression.

Apple Valsa canker is one of the most serious diseases, having caused significant apple yield and economic loss in China. However, there is still no effective biological methods for controlling this disease. Our present study focused on the inhibitory activity and mechanisms of Trichoderma longibrachiatum (T6) fermentation on Valsa mali that causes apple Valsa canker (AVC). Our results showed that the T6 fermentation exhibited effective antifungal activity on the mycelial growth and conidia germination of V. mali, causing mycelium malformation and the hyphal disintegrating in comparison to the control. The activity of pathogenically related enzymes that are secreted from V. mali and the expression level of gene of V. mali were significantly inhibited and downregulated by treatment with T6 fermentation. In addition, the lesion area and number of pycnidia of V. mali formed on the branches were significantly reduced after treatment with the T6 fermentation through the pathogenicity test on the detached branches. Our results indicate that the possible mechanism of T6 fermentation against V. mali occurs through inhibiting its growth and reproduction, the pathogenic enzyme activity, and its related gene expression.