Comparison Of Alternaria Isolates Causing Brown Spot, Black Rot And Symptomless On Citrus

Fungal species belonging to the Alternaria genus are ubiquitous saprophytes or endophytes. Alternaria spp. cause four diseases on citrus:Alternaria brown spot of tangerines(Citrus reticulata Blanco) and their hybrids, leaf spot of rough lemon (C. Jambhiri Lush), black rot of fruits of several Citrus spp. and mancha foliar de los citricos of Mexican lime(C. aurantiifolia (Christm.)Swingle). To gain a better understanding of the relationships among Alternaria spp. found on citrus, a total of 81 Alternaria strains were single-spore isolated from symptomatic(brown spot, black rot and necrotic lesions of citrus with unknown causes) and asymptomatic citrus tissues. Fungal isolates were randomly selected and examined for morphological and biological characteristics, pathogenicity and the presence of ACT-toxin synthesis genes. Their phylogenetic relationships were determined based on the analyses of multiple nuclear genes. The results are summarized as follows:Alternaria isolates(two from black rot, five from brown spot and two endophytes) were cultured on PCA plates. Morphological characteristics in terms of shape, color, size and diaphragm numbers of conidia, conidiophores, and the patterns of conidial chains varied greatly among test isolates regardless their origins. The results confirm further that morphology fails to distinguish different pathotypes of Alternaira spp. of citrus.Three strains, ZJMJR25(type strain of black rot), ZJOGS2(type strain of Alternaria brown spot) and FJGMW7(an endophytes) cultured on PDA, PCA and V8 plates showed no significant differences in regard to radial growth and spore production on either medium. Although the preference to use different carbon and nitrogen sources varied among test strains, no strong correlations between different pathotypes were observed. Due to limited sample sizes, more tests are warranted to draw firm conclusion.Pathogenicity tests were assayed on young leaves of Ponkan and Ougan by brushing 100μl of conidia suspension(5×105 conidia/ml) onto the upper surface of the leaf. Fungal isolates collected from brown spot induced the typical symptoms of brown spot, with significant yellow halos, resulting in disease rating scale of 4 to 5 (maximum=5). Some isolates of black rot induced much milder lesions with no visible yellow halos on young leaves of Ponkan(4/39) and Ougan(8/39). Some isolates from symptomless citrus tissues also induced mild lesions on young leaves of Ponkan (12/20) and Ougan(4/20) with disease index below 3. Pathogenicity tests were also assayed on mature fruit of Ponkan and navel by aseptically injecting 200 μl of conidia suspension(5×105conidia/ml) through the stem end of the fruit into the columella with a syringe. All test strains regardless of their origins induced black rot symptomatology on fruit of Ponkan and navel orange.The presence of the ACT toxin biosynthesis genes was examined by PCR amplification with two primer sets. The primer pairs ACTT1F2/ACTT1R2 amplified a 785-bp fragment and the pair ACTT2F1/ACTT2R1 amplified an 840-bp fragment exclusively from genomic DNA prepared from brown spot isolates. No DNA fragments were amplified with either primer pair from genomic DNA prepared from black rot isolates or endophytic strains.Phylogenetic trees were constructed independently or in combination based on partial sequences of an endopolygalacturonase gene(endoPG) and two anonymous OPA 1-3 and OPA 2-1 loci of Alternaria spp. including 75 strains in this study and 24 worldwide collected Alternaria alternata isolates. The results revealed that all test strains were separated into six different subclades, grouped with four clades. While test strains from China were separated into three clades (Clade 1,3 and 4),and there were no obvious correlations in terms of population genetics, pathogenic differentiation, geographic regions, host origins, or disease symptoms.Taken together, we propose that Alternaria alternata associated with citrus in China could be an Alternaria complex composed of different pathogenic variants. Alternaria alternata isolates causing black rot and brown spot differ primarily in host range, pathogenicity and disease epidemiology. The ACT-toxin synthesis genes resided on a conditionally dispensable chromosome(CDC) are solely present in the genome of the pathotype causing Alternaria brown spot, which could be used to distinguish black rot pathotype from brown spot pathotype. PCR amplification with primers ACTT1F2/R2 and ACTT2 F1/R1 could provide fast and reliable identification of Alternaria brown spot pathotype.