First Report of Anthracnose on Pepper Fruit Caused by Colletotrichum scovillei in Brazil.

Anthracnose is major disease of pepper (Capsicum annum) in the tropics and causes severe damage both in the field and postharvest. In Brazil, this disease is caused by Colletotrichum acutatum, C. boninense, C. capsici, C. coccodes, and C. gloeosporioides, where the first species is responsible for 70% of all occurrences (3). Recently, C. acutatum has been considered a species complex (1); thus, the aim of this study was to verify the etiology of anthracnose on peppers using a morphological and molecular approaches. In 2011, pepper fruits with typical symptoms of anthracnose (dark, sunken spots with concentric rings of orange conidial masses) were collected in Viçosa, Minas Gerais, Brazil. A single spore isolate was obtained on potato dextrose agar (PDA), and the derived culture was deposited in the Coleção de Culturas de Fungos Fitopatogênicos "Prof. Maria Menezes" (code CMM-4200). The upper side colonies on PDA were gray, cotton-like, and pale gray to pale orange. Conidia were hyaline, aseptate, smooth, straight, cylindrical with round ends or occasionally with end ± acute, 12.5 to 17 µm long and 3.5 to 4 µm wide on synthetic nutrient deficient agar. The isolate was morphologically typical of species belonging to the C. acutatum complex. Molecular identification of the pathogen was carried out and sequences of the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (ßt) were obtained and deposited in GenBank (Accession Nos. KJ541821 to KJ541823). A search in the Q-bank fungi database using the ITS, ßt, and GAPDH sequences retrieved C. scovillei with 100% identity for all three genes. This pathogen was previously reported in Capsicum spp. only in Thailand, Indonesia, and Japan (1,2). To confirm pathogenicity, drops with 105 spores/ml were deposited in 10 artificially wounded fruits (cv. Itapuã 501 and Melina). In control fruits, drops of sterilized water were deposited onto wounds. The fruits were covered for one day with a transparent plastic bag with moisture supplied by a wet filter paper. The fruits were detached and mature. The bags were removed, and the fruits were incubated for 10 days in a growth chamber at 25°C with a photoperiod of 12 h. After 4 days, gray-brown to black sunken spots with concentric rings were observed on 100% of the wounded fruits that had been inoculated. No disease was observed on the control fruits. The fungus C. scovillei was successfully re-isolated from symptomatic fruits to fulfill Koch's postulates. To our knowledge, this is the first report of anthracnose on pepper fruit caused by C. scovillei in Brazil. Due to the diversity of species that cause anthracnose in Capsicum, future studies using morphological and molecular tools are essential for the correct identification of Colletotrichum spp. on pepper in Brazil. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) T. Kanto et al. J. Gen. Plant. Pathol. 80:73, 2014. (3) M. J. Z. Pereira et al. Hortic. Bras. 29:569, 2011.

First Report of Enterobacter cowanii Causing Bacterial Spot on Mabea fistulifera, a Native Forest Species in Brazil.

In the summer of 2011, in a nursery located in Viçosa City, Minas Gerais State, brownish, necrotic, irregular spots were observed on leaves of Mabea fistulifera Mart. (Euphorbiaceae), an indigenous forest species commonly found in Brazil. Around 6,300 seedlings were evaluated and as many as 60% of them showed disease symptoms, including severe defoliation and plant death. Leaves with coalescing lesions turned papery in texture and had a blighted appearance. Bacterial colonies were isolated from these symptomatic leaves on King B's medium and identified based on biochemical and molecular analysis, as a member of the Enterobacteriaceae family. Like other members of the Enterobacteriaceae family, the bacteria were facultative anaerobic, gram-negative, cream-colored on YDC medium, urease and oxidase negative, as well as catalase and asparagine positive. Bacterial DNA was extracted from pure culture grown overnight in liquid 523 medium at 28°C using the Wizard Genomic DNA Purification kit (Promega) and conserved sequences in 16S rDNA (3) and rpoB (1) were amplified by PCR. The sequence of the 1,300-bp 16S rDNA fragment and the 750-bp rpoB gene were analyzed by NCBI BLAST. Related sequences were aligned and analyzed by ClustalW in MEGA 5 software. Phylogenetic analysis by maximum likelihood, using PAUP version 4.0 and TBR algorithm with 1,000 bootstrap replications, grouped the isolate in a clade with Enterobacter cowanii and the result showed 99% and 98% identity to the 16s rDNA and rpoB, respectively. The isolate clustered closely with the type strain of E. cowanii in both phylogenetic trees constructed. Pathogenicity tests were carried out by inoculating leaves of healthy seedlings either by spraying or cutting with a scissor previously dipped into a 108 CFU/ml bacterial suspension. The experiment was in a completely randomized design, with six replications. A pot with one plant was considered one experimental unit. Control seedlings were sprayed or cut with a scissor treated with saline solution. Prior to and after inoculation, plants were kept in a humid chamber for 24 h at 26°C in the dark and at room temperature. Subsequently, plants were transferred to growth chamber at 26°C, under a 12-h photoperiod (40 µmol/s/m2). Consistent with the symptoms observed originally, 7 days after inoculation, all seedlings developed leaf spots. No characteristic symptoms could be observed in the negative control. Furthermore, Koch's postulates were confirmed by reisolation of the bacterium from symptomatic tissues. In summary, the phenotypic, biochemical, and molecular tests identified the pathogen as E. cowanii. Recently, E. cowanii was isolated from Eucalyptus trees with symptoms of bacterial blight, although its pathogenicity was not demonstrated (2). To the best of our knowledge, this is the first report of a member of the Enterobacteriaceae family causing disease in M. fistulifera. The result has a great importance to better understand the role of E. cowanii as a pathogen-causing disease on a forest species. References: (1) C. L. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. L. Brady et al. Lett. Appl. Microbiol. 49:461, 2009. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.