ABSTRACT
Ipomoea nil (Linnaeus) Roth, belonging to the Convolvulaceae family, is an ornamental and medicinal plant in China, which has the function of diuretic and expectorant, and it is also a common weed in the field. In October 2021, a leaf spot disease was observed on I. nil in a field as weed in Jingzhou (N 30° 21', E 112° 19'), Hubei Province, China. Symptoms began as small brown blotches, then developed into oval or irregularly shaped brown necrotic lesions. In severe cases, the leaves were completely necrotic and detached. In the surveyed area, the incidence was between 30% - 40%. To isolate the pathogen, twenty-one leaf pieces (5×5 mm) were cut from the lesion edges of seven symptomatic leaves, disinfected with 70% ethanol and 2% sodium hypochlorite (NaOCl), rinsed with sterile water five times, then placed on three potato dextrose agar (PDA) modified with 50 µg/mL kanamycin, and incubated at 25 °C in dark for 5 days. The isolates were subcultured by transferring mycelium tips. Sixteen fungal strains were isolated from the tissues, and nine of them showed similar morphological characteristics. After cultured 7 days on PDA at 25 °C, the nine colonies were initially white, then turned greenish brown to black in the center and had abundant fine villous aerial mycelia up to 61.5 mm in average diameter. To examine its conidial morphology, the fungi were cultured for 7 days on potato carrot agar (PCA) at 22°C with a light/dark period of 8/16 h. On PCA, conidia were brown or olive-brown, obclavate to obpyriform, with a short beak, one to five transverse and zero to three longitudinal septa. They formed chains of 1 - 8 conidia, with branches. Conidia were 16 - 46 µm long and 8 - 14 µm wide (n=50). These morphological features were similar to those described in Alternaria spp. (Simmons 2007). A single isolate "Q2" was selected for molecular identification because it was the most aggressive in preliminary leaf pathogenicity assays. The internal transcribed spacer (ITS) region of rDNA and histone 3 (H3) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990) and H3-1a/H3-1b (Zheng et al. 2015). BLAST analysis revealed that the sequences (ITS, ON360984; H3, ON375577) were 100% identical to Alternaria alternata (ITS, MK396607; H3, MN840996), respectively. Maximum likelihood analysis based on combined two gene sequences was conducted with an evolutionary model of GTR+I+G under 1000 bootstrap replicates. Phylogenetic tree showed that Q2 and Alternaria alternata 21-5 and BLH-YB-11 located in one clade supported with 99% bootstrap values. The pathogen was identified as A. alternata. To fulfill Koch's postulate, 10 ml conidia (106 spores/ml) of Q2 was sprayed on five healthy seedlings, with sterile distilled water as a control. All leaves were rinsed three times with sterile water before inoculation. All seedlings were placed in sealed plastic bags with air valves, and grown in a greenhouse (25 ± 2 ËC, RH 65%). The test was repeated twice. After 10 days, symptoms typical of brown blotches similar to those observed in the field were observed on leaves of inoculated plants, while control remained healthy. A. alternata was re-isolated from the inoculated symptomatic leaves with a frequency of 100% based on morphological and molecular characters, thus Koch's postulate was confirmed. To the best of our knowledge, this is the first report of A. alternata causing leaf spot on I. nil in China. Our findings extended the host range of the pathogen A. alternata on characteristic plants.
ABSTRACT
Bacillus amyloliquefaciens strain EA19 is an endophyte isolated from Erigeron annuus with antifungal activity against Blumeria graminis f. sp. tritici, Magnaporthe oryzae, and Fusarium graminearum. The genome sequence of this strain is 3.96 Mb and contains 3,421 coding sequences, which will facilitate an understanding of the mechanisms of biocontrol.
ABSTRACT
Conidia of the obligate biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) play a vital role in its survival and rapid dispersal. However, little is known about the genetic basis for its asexual reproduction. To uncover the primary metabolic and regulatory events during conidiation, we sequenced the transcriptome of Bgt epiphytic structures at 3 (vegetative hyphae growth), 4 (foot cells initiation), and 5 (conidiophore erection) days post-inoculation (dpi). RNA-seq analyses identified 556 and 404 (combined 685) differentially expressed genes (DEGs) at 4 and 5 dpi compared with their expression levels at 3 dpi, respectively. We found that several genes involved in the conversion from a variety of sugars to glucose, glycolysis, the tricarboxylic acid cycle (TAC), the electron transport chain (ETC), and unsaturated fatty acid oxidation were activated during conidiation, suggesting that more energy supply is required during this process. Moreover, we found that glucose was converted into glycogen, which was accumulated in developing conidiophores, indicating that it could be the primary energy storage molecule in Bgt conidia. Clustering for the expression profiles of 91 regulatory genes showed that calcium (Ca2+), H2O2, and phosphoinositide (PIP) signaling were involved in Bgt conidiation. Furthermore, a strong accumulation of H2O2 in developing conidiophores was detected. Application of EGTA, a Ca2+ chelator, and trifluoperazine dihydrochloride (TFP), a calmodulin (CaM) antagonist, markedly suppressed the generation of H2O2, affected foot cell and conidiophore development and reduced conidia production significantly. These results suggest that Ca2+ and H2O2 signaling play important roles in conidiogenesis and a crosslink between them is present. In addition to some conidiation-related orthologs known in other fungi, such as the velvet complex components, we identified several other novel B. graminis-specific genes that have not been previously found to be implicated in fungal conidiation, reflecting a unique molecular mechanism underlying asexual development of cereal powdery mildews.
