Biological control of Botrytis cinerea on tomato plants using Streptomyces ahygroscopicus strain CK-15.

UNLABELLED: We developed a real-time PCR assay to specifically detect and quantify the efficacy of a biological fungicide from Streptomyces ahygroscopicus var. wuyiensis on tomato leaves. This fungicide, the natural secondary metabolite wuyiencin, is an antifungal agent against Botrytis cinerea. Specific primers were designed based on the ß-actin gene sequences, which were used to detect a 303 bp fragment from B. cinerea isolates. Our assay is highly sensitive and can be used to reliably detect and quantify as little as 1·75 pg of B. cinerea DNA. We used this detection method to monitor the progression of B. cinerea infection in inoculated plant material under preventive (wuyiencin) and nonpreventive treatment. After 5 days, plants under preventive treatment exhibited a sharp decrease in fungal biomass and no symptoms, whereas plants under nonpreventive treatment displayed severe disease symptoms. The results demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of Streptomyces wuyiensis CK-15 on B. cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: Botrytis cinerea commonly produces latent or nonsymptomatic infection on and within plant tissues, which can develop into symptomatic infection when triggered by changes in environmental conditions or host plant physiology. In this study, we develop a specific, sensitive real-time PCR assay for detecting and quantifying B. cinerea on tomato leaves to determine the control efficacy of Streptomyces ahygroscopicus var. wuyiensis as a biological fungicide. Our findings demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of biological fungicides on plant pathogens.

First Report of Tomato mosaic virus Infecting Pepino in China.

Pepino (Solanum muricatum L.) is a vegetatively propagated plant that is native to South America and is commercially grown in many countries (4) including China for its juicy and fragrant fruits. An unusual disease of pepino characterized by a yield reduction of fruits and mosaic, puckering, and distortion of leaves was observed in surveys conducted during 2010 to 2011 in pepino growing regions of Gansu, Jilin, and Yunnan provinces. These symptoms were similar to a disease caused by the Tomato mosaic virus (ToMV) first reported in Spain in 1998 (3). The sap from infected pepino was mechanically inoculated to 12 plants each of Chenopodium quinoa, Lycopersicon esculentum, and Gomphrena globosa, and the resulting symptoms were similar to those incited by ToMV (1). Symptoms included yellowish local lesions on inoculated leaves of C. quinoa and systemic mosaic in L. esculentum. Necrotic or semi-necrotic local lesions developed on inoculated leaves of G. globosa followed by systemic mosaic 7 dpi. Symptomatic pepino and indicator plants were tested for the presence of ToMV using commercial double-antibody sandwich-ELISA diagnostic kits (Agdia, Elkhart, IN). The virus incidence ranged from 33.3 to 75% in pepino and the other three indicator plants. To further confirm the presence of ToMV, ELISA-positive samples were subjected to total RNA extraction using Trizol (Invitrogen) followed by reverse transcription (RT)-PCR with ToMV-specific forward (5'-ATGTCTTACTCAATCACTTC-3') and reverse primers (5'-TTAA(G)GAT(C) GCA(T)GGTGCAG(C)AGG-3'), designed to amplify a 480-bp fragment of the coat protein. Amplicons of the expected size were obtained from all ELISA-positive samples, but not from healthy pepino plants. The amplicons were cloned into the pMD18-T (TaKaRa, Da Lian, China) vector and transformed into E. coli DH-5α competent cells. The sequences obtained (GenBank Accession Nos. JX025562, JX025563, JX025565, and JX025566) shared 99.58 to 99.79% similarity with the ToMV reference sequence (Accession No. NC002692). To our knowledge, this is the first report of ToMV infecting pepino in China. ToMV is an important pathogen that is mechanically transmitted with high efficiency as a result of agricultural practices. The disease is difficult to control once infection occurs in the field. This disease has caused serious economic loss in Spain (2), thus it is important to survey and monitor the incidence and distribution of ToMV in China. References: (1) I. Kamenova et al. Acta Hort. 722:277, 2006. (2) L. Pérez-Benlloch et al. Euphytica. 120:247, 2001. (3) J. Prohens et al. Plant Dis. 82:1281, 1998. (4) J. Prohens et al. Acta Hort. 523:53, 2000.