First Report of Colletotrichum aenigma Causing Anthracnose on Bromus japonicus in Tianjin, China.

Japanese brome (Bromus japonicus Thunb.) is a weed commonly found in roadsides, floodplain wetlands, and farmlands. During September 2020 and 2021, a leaf spot disease was observed on B. japonicus in greenhouses of Baodi district, Tianjin, China (117°15'E, 39°47'N). More than 10% of the weeds were infected. Initial irregular brown spots on leaf apices continued to expand until adjacent spots coalesced. Eventually, severely infected leaves became yellow, thinner, drier and withered. Small patches (3×3 mm) were cut from symptomatic leaves, sterilized with 75% ethanol for 30s, rinsed three times with sterile water and incubated on Petri dishes with potato dextrose agar at 25°C in darkness for 7 days. Three isolates, with uniform morphology were selected for further analysis. Colonies were cottony with entire edges and aerial white mycelia; and average growth rate of 4.5 mm/day. The upper side was pale white, and the reverse side was grayish-green. Conidia were aseptate, hyaline, subcylindrical with rounded ends, 8.6 to 18.7×4.4 to 8.3 µm (n = 50). Appressoria were dark brown, oval or irregular shaped with a few lobes, 5.7 to 9.4×4.5 to 7.8 µm (n = 50). Total genomic DNA of isolates was extracted with Fungal DNA Kit (GBCBIO, Guangzhou, China). Primers for sequences of internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), and calmodulin (CAL) genes were amplified and sequenced (Weir et al., 2012). After aligned and trimmed, the sequences of TJBDA1, TJBDA2, and TJBDA3 were identical. TJBDA1 representative isolate sequences were deposited in GenBank ITS OP247554 with 99.83% (576/577) similarity to MT476809, GAPDH OP414834 with 99.59% (244/245) similarity to MT501009, TUB2 OP414836 with 100% (703/703) to MT501053, and CAL OP414835 with 100% (601/601) to MT500921. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from B. japonicus were closely related to C. aenigma with 99% bootstrap support. Pathogenicity tests were conducted on 3-leaf stage B. japonicus seedlings. Conidial suspension of TJBDA1 (1×106 conidia/ml) brushed from a 7-day-old culture of the fungus were sprayed on 9 B. japonicus seedlings. Control plants were sprayed with sterile water. All treatments were replicated four times. The treatment plants were placed in an incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms resembling ones in the fields were observed on inoculated leaves after 7 days, but control leaves remained symptomless. The fungi reisolated from diseased leaves were morphologically and molecularly identical to the inoculated isolatescompleting Koch's postulates. According to morphological, pathological characteristics and multilocus phylogenetic analysis, the isolated strains from B. japonicus were identified as C. aenigma. To our knowledge, this is a new host record for C. aenigma causing anthracnose on B. japonicus in China. Currently, B. japonicus has evolved a high level of resistance to herbicides in some regions of China (Li et al, 2022) and C. aenigma caused serious disease to B. japonicus. We hope to discover a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. aenigma.

First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 µm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 µm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and ß-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.

Disruption of Aokap6 near the kojic acid gene cluster affects the growth and kojic acid production in Aspergillus oryzae.

The kojic acid gene cluster of Aspergillus oryzae plays a key role in kojic acid synthesis. Although the kojic acid gene cluster has been found in 2010, there is little information on the function of the genes located near the kojic acid gene cluster of A. oryzae and whether these genes affect the kojic acid gene cluster containing kojA, kojR and kojT. Here, Aokap6 near the kojic acid gene cluster of A. oryzae was identified and characterized. The Aokap6 disrupted mutants were constructed by the CRISPR/Cas9 system, which exhibited increased mycelium growth and conidial formation. Disruption of Aokap6 enhanced the tolerance to cell wall, oxidative and heat stress but not osmotic stress. Deletion of Aokap6 repressed kojic acid production, together with the reduced expression of kojA, kojR and kojT. Meanwhile, knockout of kojA, kojR and kojT led to the declined expression of Aokap6, indicating that Aokap6 is required for kojic acid production in coordination with kojA, kojR and kojT. Furthermore, overexpression of kojA, kojR and kojT had no effects on the transcript level of Aokap6, and overexpression of kojA in Aokap6 deletion strain could rescue the reduced yield of kojic acid, suggesting that Aokap6 is involved in kojic acid synthesis acting upstream of kojA. These findings provide new insight for the further understanding of kojic acid gene cluster and kojic acid production in A. oryzae.

Metabolism-Based Herbicide Resistance to Mesosulfuron-methyl and Identification of Candidate Genes in Bromus japonicus.

The evolved resistance of Bromus japonicus Houtt. to ALS-inhibiting herbicides is well established. Previous studies have primarily focused on target-site resistance; however, non-target-site resistance has not been well characterized. This investigation demonstrated that ALS gene sequencing did not detect any previously known resistance mutations in a mesosulfuron-methyl-resistant (MR) population, and notably, treatment with the P450 monooxygenase (P450) inhibitor malathion markedly heightened susceptibility to mesosulfuron-methyl. Utilizing UPLC-MS/MS analysis confirmed elevated mesosulfuron-methyl metabolism in MR plants. The integration of Isoform Sequencing (Iso-Seq) and RNA Sequencing (RNA-Seq) facilitated the identification of candidate genes associated with non-target sites in a subpopulation with two generations of herbicide selection. Through qRT-PCR analysis, 21 differentially expressed genes were characterized, and among these, 10 genes (comprising three P450s, two glutathione S-transferases, one glycosyltransferase, two ATP-binding cassette transporters, one oxidase, and one hydrolase) exhibited constitutive upregulation in resistant plants. Our findings substantiated that increased herbicide metabolism is a driving force behind mesosulfuron-methyl resistance in this B. japonicus population.

Sucrose triggers a novel signaling cascade promoting Bacillus subtilis rhizosphere colonization.

Beneficial rhizobacteria promote plant growth and protect plants against phytopathogens. Effective colonization on plant roots is critical for the rhizobacteria to exert beneficial activities. How bacteria migrate swiftly in the soil of semisolid or solid nature remains unclear. Here we report that sucrose, a disaccharide ubiquitously deployed by photosynthetic plants for fixed carbon transport and storage, and abundantly secreted from plant roots, promotes solid surface motility (SSM) and root colonization by Bacillus subtilis through a previously uncharacterized mechanism. Sucrose induces robust SSM by triggering a signaling cascade, first through extracellular synthesis of polymeric levan, which in turn stimulates strong production of surfactin and hyper-flagellation of the cells. B. subtilis poorly colonizes the roots of Arabidopsis thaliana mutants deficient in root-exudation of sucrose, while exogenously added sucrose selectively shapes the rhizomicrobiome associated with the tomato plant roots, promoting specifically bacilli and pseudomonad. We propose that sucrose activates a signaling cascade to trigger SSM and promote rhizosphere colonization by B. subtilis. Our findings also suggest a practicable approach to boost prevalence of beneficial Bacillus species in plant protection.