Characterization of Alternaria Species Associated with Watermelon Leaf Blight in Beijing Municipality of China

Home , Alternaria, Alternaria alternata, Alternaria tenuissima

Journal of Plant Pathology (2016), 98 (1), 135-138 Edizioni ETS Pisa, 2015 135

Short Communication

CHARACTERIZATION OF ALTERNARIA SPECIES ASSOCIATED WITH WATERMELON LEAF BLIGHT IN BEIJING MUNICIPALITY OF CHINA

J. Zhao, S.W. Bao, G.P. Ma and X.H. Wu

Department of Plant Pathology, China Agricultural University, Beijing 100193, China

SUMMARY In addition, no further information pertaining to the presence of Alternaria species on watermelon is available in Leaf blight symptoms consisting of dark brown lesions Beijing municipality of China. The aim of this study was were observed on watermelon plants in seven districts or to investigate the Alternaria populations associated with counties in Beijing municipality of China. A total of 64 diseased watermelon leaves. Alternaria isolates were recovered and identified based on To determine the causal agents of the leaf lesions, fungi morphological characters and sequence analyses of rDNA- isolates with morphological characters similar to the de- ITS regions and histone 3 genes. Amongst these isolates, scription of Alternaria species (Simmons, 2007) were re- Alternaria tenuissima was the most prevalent species rep- covered from the diseased leaves using tissue separation resenting 76.6% of the isolates, followed by A. alternata methods (Zheng et al., 2015). Pure cultures of the isolates (23.4%). All isolates were divided into individual clades were obtained by single spore purification and were stored representing two species, A. tenuissima and A. alternata, on potato dextrose agar (PDA) slants. Colony morphology according to phylogenetic analysis of histone 3 gene. Rep- observation was conducted by incubation of the isolates on resentative Alternaria isolates of the two species induced PDA plates at 25°C in darkness for 7 days (Andersen et al., dark brown lesions on detached watermelon leaves, with 2001). Conidia and sporulation patterns were determined disease incidence ranging from 44.4-83.3%. These results on potato carrot agar (PCA) plates kept 40 cm below cool allowed the identification of the presence of Alternaria white fluorescent bulbs with 8 h/16 h periods of light/dark populations causing watermelon leaf blight for the first (Andersen et al., 2001). time in Beijing municipality of China. Genomic DNA of each Alternaria isolate was extracted using a standard phenol-chloroform extraction method Key words: leaf blight, Alternaria tenuissima, A. alter- (Lee and Taylor, 1990). The primers ITS1 / ITS4 (White nata, watermelon, histone 3 gene et al., 1990) were used for amplification of the internal transcribed spacer region of ribosomal DNA (rDNA-ITS), and primers H3-1a / H3-1b for a part of the histone 3 gene (Glass and Donaldson, 1995). Polymerase chain reaction (PCR) amplification was done according to the primer Leaf blight is one of the most commonly occurred dis- concentrations and the PCR protocols as described in our eases on watermelon (Citrullus lanatus (Thunb.) Matsum. former study (Zheng et al., 2015). et Nakai), and leads to considerable economic losses to Purification and ligation of the PCR amplification producers (Chopra et al., 1974). Alternaria cucumerina has products, as well as transformation of the ligation reac- been reported as the prevalent species causing watermelon tion mixtures were conducted according to Zheng et al. leaf blight in the world including China (Jackson and We- (2015). White colonies with the target DNA insertion ber, 1959; Kim et al., 1994; Liu et al., 2010). A. alternata verified by PCR were sent to Beijing Sunbiotech Co. Ltd. was also frequently isolated from watermelon leaves during (Beijing, China) for sequencing. Sequence alignment was the development of the disease in the field (Maheswari done using Clustal W program (version 1.83) (Thompson and Sankaralingam, 2010). Besides the dominant species et al., 1994) with manual adjustment. Phylogenetic analysis A. cucumerina, A. alternata and A. tenuissima were both was carried out by MEGA 5 program version 5.2.2 (http:// referred to cause foliar diseases on watermelon in Zheji- www.megasoftware.net/) using the neighbor-joining (NJ) ang province of China about twenty years ago; however, method. intense study on the identification and pathogenicity of the Pathogenicity test was conducted on detached fungi were not particularly conducted (Chen et al., 1993). leaves from 40-day-old plants of watermelon cv. ‘Chaoyuemengxiang’ using eight representative isolates. Corresponding author: X.H. Wu Fax: +86.10.62813785 Inoculations were made on the upper surface of each leaf E-mail: [email protected] with 20 μl of a 106 conidia/ml spore suspension (Wang et 136 Alternaria species causing watermelon leaf blight Journal of Plant Pathology (2016), 98 (1), 135-138

Table 1. Geographic origins and numbers of Alternaria isolates obtained from watermelon leaves with macroscopic leaf blight symptoms.

Alternaria species Geographical origins Number of locations Number of isolates A. tenuissima A. alternata Yanqing county 4 24 19 5 Changping district 2 8 7 1 Daxing district 4 13 11 2 Tongzhou district 4 10 7 3 Shunyi district 2 7 3 4 Miyun county 1 1 1 0 Fangshan district 1 1 1 0 In total 18 64 49 15 al., 2014; Zheng et al., 2015). After incubation for 7 days, symptoms on the inoculated leaves were recorded and compared with those occurred on the originally infected leaves. Disease severity (DS) and disease index were scored on a revised rating system of Santha et al. (2009). A total of 64 Alternaria isolates were recovered from symptomatic leaf samples of watermelon collected from seven districts or counties in Beijing municipality of Chi- na. Amongst these isolates, 49 isolates (76.6%) were identified as A. tenuissima, and the remainder 15 isolates (23.4%) were confirmed as A. alternata based on morphological traits and molecular characteristics (Table 1). Fungal isolates identified as A. tenuissima produced colonies that were gray to dark brown on PDA plates (Fig. 1A). Conidia were 23.3-36.8 × 7.9-12.2 μm in size, ovoid to obclavate in shape, with 1 to 4 transverse and 0 to 2 longitudinal septa on PCA plates (Fig. 1C). These isolates often developed long conidial chains with one or two lateral branches (Fig. 1E). Isolates identified as A. alternata had dark olivaceous colonies on PDA plates (Fig. 1B). Conidia were ovoid, ellipsoid or obpyriform with 1-4 transverse and 0-3 longitudinal septa, and measured 21.5-33.7 × 7.6- 11.8 μm (Fig. 1D). These isolates usually produced conidial chains with numerous secondary and occasionally tertiary chains on PCA plates (Fig. 1F). PCR amplification of the rDNA-ITS region gener- ated a 570-bp fragment for all the Alternaria isolates. DNA sequencing of the partial coding sequence of histone 3 gene revealed that 49 Alternaria isolates produced Fig. 1. Morphological characteristics of two Alternaria species. A-B: Colonies of the representative isolates of A. tenuissima, a 546-bp fragment, and had more than 99% similarities A. alternata on PDA plates. C-D: Conidia of A. tenuissima, A. with the sequences of the known A. tenuissima isolates alternata on PCA plates (scale bar = 50 μm). E-F: Sporulation (JX495168, KF051252) retrieved from GenBank database. patterns of A. tenuissima, A. alternata on PCA plates (scale The remainder 15 isolates obtained a 440-bp fragment, bar = 200 μm). and were 99-100% similarly to that of A. alternata isolates (KF308937, KF308912). The sequences in this study were submitted to the NCBI sequence database (https:// gene disclosed that all the 49 A. tenuissima isolates were www.ncbi.nlm.nih.gov/, accession numbers KP701052- located in one clade with the known A. tenuissima iso- KP701115 for rDNA-ITS and KP701116-KP701179 for lates from GenBank, while 15 A. alternata isolates were histone 3 gene, respectively). attributed to another clade with the existing A. alternata Phylogenetic analysis based on rDNA-ITS region was isolates (Fig. 3). difficult to differentiate between the A. tenussima and A. The inoculated watermelon leaves developed dark alternata species, since all the isolates were grouped in to brown lesions similar to those initially observed diseased one clade (Fig. 2). Phylogenetic analysis of the histone 3 leaves in the field, after incubation for 7 days (Fig. 4). Journal of Plant Pathology (2016), 98 (1), 135-138 Zhao et al. 137

Fig. 3. Phylogenetic tree constructed based on the partial coding sequences of histone 3 gene of 49 A. tenuissima iso- Fig. 2. Phylogenetic tree constructed based on the ITS re- lates, 15 A. alternata isolates, and 7 reference sequences regions of rDNA of 49 A. tenuissima isolates, 15 A. alternata iso- trieved from GenBank. lates, and 7 reference sequences retrieved from GenBank. A. cucumerina was not obtained from the diseased water- The disease incidence and disease index of watermelon melon leaves, which could be related to the limited sample leaves inoculated with A. tenuissima were 61.1-83.3% numbers and sample locations. In 1993, A. tenuissima and (72.2 ± 11.1%) and 15.28-29.17 (20.83 ± 7.35), respective- A. alternata have been mentioned to cause foliar diseases ly. The disease incidence and disease index caused by on watermelon from Zhejiang province of China (Chen A. alternata were a little lower, which were 44.4-61.1% et al., 1993). However, there has been no systematic and (53.7 ± 8.5%) and 16.67-23.61 (21.30 ± 4.01), respectively. detailed study inclined to characterization of Alternaria Although the disease incidence of watermelon leaves inoc- species on watermelon in China for about twenty years. ulated with A. tenuissima is higher than A. alternata, there These results allowed the identification of the presence of was no significant difference between the two species ac- Alternaria populations causing watermelon leaf blight for cording to the least significant difference test (P < 0.05). the first time in Beijing municipality of China. In conclusion, A. tenuissima was confirmed as the Both A. tenuissima and A. alternata were identified as dominant species causing leaf blight on watermelon fol- the causal agent of foliar diseases on sunflower (Wang et lowed by A. alternata in Beijing municipality of China. al., 2014) and potato (Zheng et al., 2015). There were also 138 Alternaria species causing watermelon leaf blight Journal of Plant Pathology (2016), 98 (1), 135-138

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Received July 21, 2015 Accepted October 10, 2015