First report of Alternaria tenuissima causing leaf black spot on pecan in China

China is one of the largest markets for pecan (Carya illinoinensis) consumption in the world, and also, pecan production in China has been increased by years since 2008 (Zhang et al. 2015). From July to September in the year 2019 and 2020, leaf black spot was observed on several pecan cultivars including Pawnee, Burkett, Kiowa and Western schley in the germplasm in Liuhe county, Nanjing, Jiangsu Province, China. Disease incidence was approximately 40% in 2019 and 50% in 2020 respectively. Small, dark brown to black spots on leaves were observed initially, and spots expanded quickly into circular or irregular when spots coalesced. In severe cases, the disease can obviously weaken the tree vigor, ultimately leading to losses in yield. Disease symptoms were not observed on the fruits. To determine the causal agent of black leaf spot, symptomatic leaves were collected and cut into pieces (approximately 3 × 3 cm2), surface sterilized with 1% sodium hypochlorite for 2 min and 75% ethanol for 30 s and rinsed twice with sterile distilled ddH2O. Dried tissues were placed on potato dextrose agar (PDA) amended with rifampin (Solarbio, Beijing, China) at a final concentration of 100 µg/mL and incubated at 25°C for 7 days in darkness. Five colonies were obtained and purified by single spore culture for morphological characterization. Colonies were initially white, turned to dark olivaceous with white margin and moderate to abundant gray aerial hyphae. Conidiophores were linear, light brown in color and appeared as individuals or in clusters. Conidia were pale brown to brown, typically obclavate or obpyriform (8 – 49 µm× 3 – 18 µm), with one to five transverse septa and zero to three longitudinal septa (n=50). Conidia of all isolates produced no beaks or a short beak. Based on conidial morphological characteristics, isolates were tentatively identified as Alternaria tenuissima (Simmons 2007). To further confirm the five isolates, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), partial region of the histone 3 (H3) gene, translation elongation factor 1-α gene (TEF) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with PCR primer sets ITS1/ITS4 (White et al. 1990), H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), and GDF1/GDR1 (Berbee et al. 1999) respectively. The sequences were deposited in GenBank (ITS, MN822659 to MN822661 and MZ182355 to MZ182356; histone 3, MN840997 to MN840999 and MZ202355 to MZ202356; TEF, MZ246595 to MZ246599; GAPDH, MZ246590 to MZ246594). BLAST analysis of the resulting sequences showed 99% to 100 % nucleotide identical to those of A. tenuissima isolates (KP278184 [ITS]; MH824352 [H3]; MN046379 [TEF]; MK683840 [GAPDH]). Therefore, based on morphological characteristics and DNA sequences data, the five isolates were identified as A. tenuissima. To determine the pathogenicity of the five isolates, 10 µL of 105 conidia /mL suspension from each isolate was placed to three intact young leaves (Pawnee) respectively. Leaves inoculated with ddH2O in the same manner served as the controls. Inoculated leaves were placed in a growth chamber at 28°C with 95% relatively humidity (RH). Black spot symptoms appeared on all inoculated leaves by 7 days post inoculation, A. tenuissima were re-isolated from the inoculated leaves with isolates and reconfirmed by morphological characteristics, thus fulfilling the Koch’s postulates. The controls remained symptomless. Pathogenicity tests were conducted twice. To our knowledge, this is the first report of A. tenuissima causing black leaf spot on pecan in China. Although we have not found any fruit infection, we think the disease is also a potential threat to pecan fruits and production. If more reports of this pathogen are found on pecans, then it is necessary to study and develop effective control strategies.

Mangrove Dieback and Leaf Disease in Sonneratia apetala and Sonneratia caseolaris in Vietnam

Even though survival rates for mangrove restoration in Vietnam have often been low, there is no information on fungal pathogens associated with mangrove decline in Vietnam. Therefore, this research was undertaken to assess the overall health of mangrove afforestation in Thanh Hoa Province and fungal pathogens associated with tree decline. From a survey of 4800 Sonneratia trees, the incidence of disorders was in the order of pink leaf spot > shoot dieback > black leaf spot for S. caseolaris and black leaf spot > shoot dieback > pink leaf spot for S. apetala. Approximately 12% of S. caseolaris trees had both pink leaf spot and shoot dieback, while only 2% of S. apetala trees had black leaf spot and shoot dieback. Stem and leaf samples were taken from symptomatic trees and fungi were cultured in vitro. From ITS4 and ITS5 analysis, four main fungal genera causing leaf spots and shoot dieback on the two Sonneratia species were identified. The most frequently isolated fungal taxa were Curvularia aff. tsudae (from black leaf spot),Neopestalotiopsis sp.1 (from stem dieback), Pestalotiopsis sp.1 (from pink leaf spot), and Pestalotiopsis sp.4a (from black leaf spot). The pathogenicity of the four isolates was assessed by under-bark inoculation of S. apetala and S. caseolaris seedlings in a nursery in Thai Binh Province. All isolates caused stem lesions, and Neopestalotiopsis sp.1 was the most pathogenic. Thus, investigation of fungal pathogens and their impact on mangrove health should be extended to other afforestation projects in the region, and options for disease management need to be developed for mangrove nurseries.

Characterization of Alternaria brassicae causing black leaf spot disease of cabbage (Brassica oleracea var. capitata) in the southern part of Kazakhstan

Cabbage plants showing symptoms of leaf spot were detected from various fields in the Almaty region of Kazakhstan in the winter seasons of 2015 and 2016. The disease incidences of approximately 50% were recorded in various fields visited in the Almaty region. The pathogen was aseptically isolated from the symptomatic leaves and maintained in an in vitro culture media. Morphological characteristics and sporulation of the fungus was determined under both light and electron microscopy. The extracted genomic DNA of the fungi was subjected to Polymerase Chain Reaction (PCR) using ABCsens/ABCrev and ITS1/ITS4 primers amplifying ABC transporter (Atr1) gene and the internal transcribed spacer regions, respectively. The amplified products of PCR were sequenced, aligned, blasted and compared for similarity with other species in the NCBI GenBank. The cluster analysis result showed 99% homology with related fungi retrieved from the NCBI GenBank for the ITS region. The fungal isolate was pathogenic towards twenty-two-day-old plants, namely, Brassica oleracea, Lycopersicon esculentum, Solanum melongena, and was established as the causal agent of leaf spot on these plants. This is the first record implicating A. brassicae for black leaf spot disease of cabbage in Kazakhstan.

Fungi associated with leaves of Sonneratia apetala Buch. Ham and Sonneratia caseolaris (L.) Engler from Rangabali coastal zone of Bangladesh

A total of six species and one genus of fungi associated with black leaf spot of Sonneratia apetala Buch. Ham (Kewra); and anthracnose and small leaf spot of S. caseolaris (L.) Engler (Choila) were isolated following “Tissue planting” method. The fungi associated with black spot of S. apetala were Aspergillus fumigatus Fresenius, Colletotrichum lindemuthianum (Sacc. & Magn.) Br. & Cav., Pestalotiopsis guepinii (Desm.) Stey. and Phoma betae Frank. Anthracnose of S. caseolaris showed the association of A. fumigatus and P. guepinii. The fungi associated with leaf spot of S. caseolaris were Curvularia fallax Boedijn, Fusarium Link, Penicillium digitatum (Pers.) Sacc. and P. betae. Frequency percentage of association of P. guepinii was highest (74.10) in black spot of S. apetala whereas the same fungus showed highest frequency percentage (85.70) in case of anthracnose of S. caseolaris. Phoma betae showed highest frequency percentage (60.00) in leaf spot of S. caseolaris. Phoma betae is first time recorded from Bangladesh. Dhaka Univ. J. Biol. Sci. 27(2): 155-162, 2018 (July)

Validation of Inoculation test and Screening for Resistance Sources of Mustard-Rapeseed Cultivars in Bangladesh Against Alternaria brassicicola

Alternaria leaf spot or black leaf spot caused by Alternaria brassicicola is the devastating diseases of mustard-rapeseed in Bangladesh, and can cause yield loss up to 60% every year. A total of 27 cultivars of Brassica spp. where Brassica rapa (13), B. juncea (5) and B. napus (9) were selected for resistance screening against A. brassicicola. Two inoculation methods viz. detached leaf and seedling inoculation were assessed for checking the validation of inoculation technique using cultivated mustard-rapeseed varieties in Bangladesh a method to measure resistance to A. brassicicola. A significant positive correlation between the results of two inoculation methods was found in this study. The detached leaf technique was more suitable due to development of clear symptoms on the leaves within 36 hrs, and suitable for screening large scale genotypes for resistance. The 3rd leaves of 30 day-old were more suitable for inoculation having severe symptoms than the 4th leaves. Among 27 Bangladeshi mustard-rapeseed cultivars all cultivars expressed susceptible reaction to A. brassicicola, except BINA Sharisha-8, as it had border line resistance.