Walnut (Juglans sinensis L.) is an important economic tree. Its fruit are rich in omega-3 essential fatty acids, which are valuable nutritionally (Cheon et al, 2013). In March 2019, severe branch blight of walnuts (cv. Qingxiang) were observed in two fields in Ganquan Town, Gansu Province, China (N 33°56'/E105°44'). The incidence was about 3% among 10,000 walnuts. Disease symptoms included fusiform or oval black lesions gradually expanded on the branches, blight and dieback of branches, reddish brown dead branch bark with lots of black small spots (pycnidia), and defoliation. To isolate pathogen, 30 diseased tissues collected from different trees were disinfected with 0.1% HgCl solution for 1 min, rinsed in sterilized water 3 times, and placed on potato dextrose agar (PDA) at 25℃ in the dark. About three days later, 4 fungal isolates (3-3, 3-6, 3-9 and H3) with similar morphological characteristics were obtained. Their colonies, with regular margin on OA, 6.1~6.8 cm diam. after 7 days, were loose, greenish olivaceous to olivaceous grey, velvety, floccose to woolly. The conidia (n=60) were 4.77 to 8.84 μm long (mean = 6.88 μm; SD ± 0.91 μm) × 1.71 to 3.89 μm wide (mean = 2.81 μm; SD ± 0.53 μm), cylindrical, ellipsoidal to oblong, hyaline and aseptate. Pycnidia (n=25) were 76.66 ~ 132.86μm diam. (mean = 102.93 μm; SD ± 12.15 μm), variable in shape and size, mostly globose to subglobose. These characteristics were similar to B. exigua var. exigua (Boeremia et al, 2004). Pathogenicity tests of four isolates were performed 3 times on 5 healthy 2-3 years old walnuts (cv. Qingxiang). Plants were wounded by insect needle No.6 and inoculated with 5 mm mycelium block grew on PDA medium or PDA medium as control and kept moist in climatic cabinet (> 85% RH, 25°C). After 3 days, oval black lesions were occurred on branches and gradually expanded, but control was asymptomatic. And original isolates were re-isolated from these diseased shoots. Genomic DNA of four isolates were extracted, and the internal transcribed spacer (ITS), β-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2) gene were amplified and sequenced using the primers ITS1/ITS4, Btub2Fd/Btub4Rd and RPB2-5F2/fRPB2-7CR (White et al, 1990; Woudenberg et al, 2009; Chen et al, 2015), respectively. Sequences were deposited in GenBank (accession no. ITS: MT154621, MT154622, MT154623, MT154624; tub2: MT223481, MT223482, MT223483, MT223484; rpb2: MW448152, MW448153, MW459982, MW459983), and compared with available sequences in NCBI. Results showed that ITS of four isolates have 100% sequence identity to Boeremia spp., tub2 and rpb2 have 100% sequence identity to B. exigua var. exigua (GenBank accession no. MN983734, MN983315) and B. exigua var. linicola (GenBank accession no. MN983785, MT920619). According to host specificity (Boeremia et al, 1976). A 106 conidium/mL spore suspension of four isolates or sterile water were inoculated on stem base of two-month old flax seedlings, placed in climatic cabinet (> 85% RH, 25°C) for moisturing and repeated three times. After two weeks, all inoculated plants still were asymptomatic, indicated that four isolates aren’t B. exigua var. linicola. Therefore, they were identified as B. exigua var. exigua based on morphological characteristics, molecular analysis and pathogenicity tests. To our knowledge, this is the first report of B. exigua var. exigua causing walnut branch blight worldwide, which will provide further guidance for prevention and control of walnut branch blight.
Vitamins, capsaicin and capsochrome are abundant in pepper (Capsicum annuum), a fruit that is also used as a spice. During hot and rainy seasons, anthracnose disease caused by Colletotrichum spp. affects pepper crops and causes significant yield losses in the pre- and post-harvest stages(Liu et al. 2016). Unidentified disease spots were discovered on peppers leaves in a field in Wei yuan (35°8'10" N, 104°12'54" E), Gansu Province, China, in September 2019. The diseases was found to have a 100% incidence in a 0.07-ha area, which drew our attention. The lesions were mostly found in the middle and upper parts of the leaves, and the symptoms mostly showed up as roughly circular patches on the leaves with dark brown, and yellowish center. 18 tissues with a diameter of 1 cm were obtained from the line between healthy and diseased portions. They were sterilized for 45 s in 1% mercuric chloride, then rinsed 5 times in sterile distilled water and dried with sterile filter paper. After 4 days of culture on a plate with a PDA media 5 strains were recovered from the treated tissue. Healthy pepper plants grown in the lab were inoculated with conidia suspension (50 mL, 107 conidia/mL) for pathogenicity while sterile distilled water was used as control. Each treatment had three duplicates. Leaves infected with the BYL strain 16 days later showed obvious symptoms, which were comparable to those found in the field. The control leaves showed no sign of disease. The pathogen was re-isolated from the infected pepper leaves and it had the same features as strain BYL. Koch's postulate was proven correct. The BYL colony started out white, then turned gray-brown with black sclerotia in the center. Conidia were hyaline, smooth, cylindrical, typically straight, with rounded ends, and ranged in size from11.754-16.477(14.587±0.139×2.833-4.220(3.348±0.037) μm. Appressoria solitary or in loose clusters, 6.910-9.078×5.386-7.119 μm in size, medium brown, smooth-walled, ellipsoidal or irregular in form, with noticeable piercing pore with dark halo. The isolate was identified as Colletotrichum species based on the morphological characteristics (Damm et al. 2014).It was then re-identified using multi-molecular analysis. To amplify and sequence of the isolates, the genes ITS, TUB2, CHS1, ACT, GAPDH and HIS3 were employed (Weir et al. 2012, Crous et al. 2004). They were deposited in GenBank (MW581857 for ITS, MW595706 for ACT, MW595707 for CHS1, MW595708 for GAPDH, MW595709 for HIS3, and MW595710 for TUB2). The sequence of ITS, ACT, CHS1, and HIS3 in GenBank were found to be 100% identical to those of Colletotrichum tabaci (JQ005763 for ITS, KM105414 for ACT, JQ005784 for CHS1 and KM105346 for HIS3). The primers GAPDH and TUB2 amplified a gene sequence that was 99% identical to Colletotrichum tabaci in GenBank (KM105559 for GAPDH and JQ005847 for TUB2). Based on appearance and sequencing analysis, the isolate was identified as Colletotrichum tabaci. The optimal light condition for BYL growth was 12 h light/12 h dark cycle, temperature 30 o C, pH 8, sucrose as carbon source, and yeast extract as nitrogen source according to the biological features. Colletotrichum tabaci caused anthracnose in peppers in the field. This is the first report of Colletotrichum tabaci causing anthracnose in peppers in China that we are aware of.
Fennel (Foeniculum vulgare Mill.) is herbaceous plant commonly cultivated for culinary and medicinal uses in China (Shi et al. 2016 ). In May 2019, disease of fennel was observed in Yumen City, Gansu Province, China (N 40°28'/E 97°05'). The incidence across the fields (about 0.23 hectare) was about 4.5%. The outer leaves of diseased fennel wilted, the rhizome changed color from brown to dark brown,necrosis and rot symptoms developed on the root. Finally, the whole plant wilted and died. When pulling up, it was easy to break the root. To identify the pathogen, 15 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl solution for 30 s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃ in the dark. About 7 days, hyphal tips from the tissue edge were transferred to a new PDA for purification. Three isolates obtained were named as hxa, hxb and hxc. To confirm their pathogenicity, two-month old fennel seedings planted in pots, three seedings per pot, with sterilized nutrient soil were inoculated by pouring 50 ml of conidial suspension (107 conidium/mL) produced from the three isolates. Plants inoculated with sterilized water only were included as controls. Six pots of inoculated plants were maintained in climatic cabinet / chamber (> 85% RH, 25°C). The pathogenicity tests were conducted twice. After 7 days, the plants inoculated with conidial suspension of hxa developed brown necrosis and wilt symptoms resembling those originally observed in the field, whereas the controls and the plants inoculated with the other two isolates had no symptoms. Furthermore, hxa was reisolated from rhizome of these diseased plants. The results indicated that isolate hxa was the pathogen causing root rot of fennel. The colonies of hxa on PDA were white-to-cream, slimy, mycelium appressed, aerial mycelium absent. Mycelium was hyaline, septate and formed hyphal coils. Conidiophores were solitary, hyaline, sometimes crooked or sinuous, widest at the base, gradually tapering to the apex. Conidia were smooth, hyaline, aseptate, elliptical and ovoid, measuring 5.97 to 9.51 × 2.13 to 3.58 um (avg. 7.58×2.78, n=100). These morphological characters of the fungal isolates were identical to those of Plectosphaerella cucumerina (Carlucci et al. 2012). For molecular identification, genomic DNA was extracted from the mycelium, and the rDNA internal transcribed spacer (ITS) region, portions of the 28S ribosomal RNA (LSU), calmodulin (CaM) and translation elongation factor 1α (Ef‐1α) gene were amplified using primer pairs ITS1/ITS4, LROR/LR5, CMD5/CMD6 and 688f/1251R (White et al., 1990; Hopple et al., 1999; Hong et al., 2005; Alves et al., 2008), respectively. The sequences of these genes were deposited in GenBank (accessions: ITS as MW426266, LSU as MW433724, CaM as MW448071 and EF-1a as MW459981) and used in analysis to generate a phylogenetic tree. These sequences showed 100, 100, 96 and 97.32% homology to the sequences of P. cucumerina (GenBank accession no. EU594566, MH867359, KY416911 and KY964491), respectively. According to morphological characteristics and phylogenetic analysis, isolate hxa was identified as P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing root rot of fennel in China as well as worldwide. This finding may help to take effective control measures of root rot on fennel.
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