Studies On Molecular Detection And Identification Of Botrytis Species Infecting Major Fruits And Vegetables

The gray mold diseases are caused by Botrytis spp. and Botrytis-like fungi. Use of molecular detection methods to identify species of Botrytis and related fungi is a convenient way for monitoring the epidemics of the gray mold diseases. Previous research already identified some species of Botrytis and Botrytis-like fungi. But we need a detection and identification system. Therefore, a study was done to develop species-specific PCR primers and to establish PCR assays to identify Botrytis species on broadbean, table grapes and Allium crops.1. PCR-based assays to detect and differentiate three Botrytis species(B. cinerea, B. fabae and B. fabiopsis) on broadbean were established. Two sets of PCR primers, Bc-f/Bc-r and Bfab-f/Bfab-r for B. cinerea and B. fabiopsis, respectively, were designed based on two SCAR markers derived from two RAPD assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the NEP1 gene sequence(NEP1 coding for necrosis and ethylene-inducing protein 1). The three primer sets were highly specific for the corresponding species of Botrytis both in single and multiplex PCR assays. The PCR detection limit was 40 pg, 40 pg and 400 pg DNA per 25-μL reaction mixture for B. fabae, B. fabiopsis and B. cinerea, respectively. Presence of the broadbean DNA in the PCR reactions at 1:1000(Botrytis DNA: broad bean DNA, w/w) had negligible effects on detection of the targeted Botrytis species. The multiplex PCR assay was able to detect three Botrytis species in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to detect and differentiate Botrytis species causing chocolate spot of broad bean.2. PCR-based assays to detect and differentiate four Botrytis species on table grapes(B. cinerea, B. pseudocinerea, B. sinoviticola and Botrytis sp.2) were developed. Four sets of primers, Bc-f/Bc-r for B. cinerea, Bps-f/Bps-r for B. pseudocinerea, Bsa-f/Bsa-r for B. sinoviticola and Bsp2-f/Bsp2-r for Botrytis sp.2, were designed based on four SCAR markers derived from three RAPD assays. The four primer sets were highly specific for the corresponding species of Botrytis in single PCR assays. The PCR detection limit was 0.7 ng, 0.7 ng, 0.7 ng and 400 pg DNA per 25-μL reaction mixture for B. pseudocinerea, B. sinoviticola, Botrytis sp.2 and B. cinerea, respectively. The single PCR assay was able to detect four Botrytis species in artificially infected grape leaves.3. PCR-based assays to detect and differentiate six Botrytis species on Allium crops(B. aclada, B. byssoidea, B. cinerea, B. porri, B. squamosa and B. sinoallii) were developed. Six sets of primers, Bc-f/Bc-r for B. cinerea, Bac-f/Bac-r for B. aclada, Bby-f/Bby-r for B. byssoidea, Bpo-f/Bpo-r for B. porri, Bsq-f/Bsq-r for B. squamosa and Bsi-f/Bsi-r for B. sinoallii, were designed based on six SCAR markers derived from four RAPD assays. The six primer sets were highly specific for the corresponding species of Botrytis in single PCR assays. The PCR detection limit was 0.07 ng, 0.07 ng, 7 pg, 7 pg, 7 pg and 400 pg DNA per 25-μL reaction mixture for B. aclada, B. byssoide, B. porri, B. sinoallii, B. squamosa and B. cinerea, respectively. The single PCR assay was able to detect six Botrytis species in artificially infected allium crops leaves.4. The DNA sequences of ITS and three nuclear genes(G3PDH, HSP60 and RPB2) were tested with 63 isolates of 32 species of Botrytis and Botrytis-related genera to investigate feasibility of using these DNA sequences as Botrytis DNA barcoding. The investigated fungal isolates were pairwise compared for interspecific and intraspecific differences in the sequences of ITS or each nuclear gene. A neighbor-joining tree was generated based on the DNA sequences of the three nuclear genes singly. The results showed that among ITS and the three nuclear genes, the partial sequences of G3 PDH gene appeared to a proper candidate used as DNA barcoding. A PCR primer set, namely G31-f/G31-r, was designed. It could generate DNA fragments sized by 789 bp to 798 bp. The PCR amplified DNA sequences were confirmed to be able to differentiate Botrytis species and Botrytis-related fungi.In this study, we got species-specific primer sets and designed the DNA barcoding for detection and identification of Botrytis species. These results will be useful for further study of the population biology and epidemics of the gray mold fungi.