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ARS Home » Pacific West Area » Pullman, Washington » Grain Legume Genetics Physiology Research » Research » Publications at this Location » Publication #314767
Title: Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean

Author
item FAN, XUAN - Huazhong Agricultural University
item ZHANG, JING - Huazhong Agricultural University
item YANG, LONG - Huazhong Agricultural University
item WU, MINGDE - Huazhong Agricultural University
item Chen, Weidong
item LI, GUOQING - Huazhong Agricultural University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2014
Publication Date: 5/1/2015
Citation: Fan, X., Zhang, J., Yang, L., Wu, M., Chen, W., Li, G. 2015. Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean. Plant Disease. 99:691-698.

Interpretive Summary: The grain legume faba bean (broad bean) is an important pulse crop worldwide. It suffers a devastating chocolate spot disease which could be caused by three morphologically similar species of Botrytis (B. cinerea, B. fabae and B. fabiopsis). However, the three species have different temperature preferences and fungicide sensitivities. Thus it is important to identify the species in disease diagnosis in order to implement appropriate control practices. Two sets of PCR primers specific for B. fabiopsis were designed based on two SCAR markers . Another PCR primer set specific for B. fabae was designed based on a protein gene sequence. With the existing specific PCR primers for B. cinerea, these primer sets were highly specific for the corresponding species of Botrytis both in single and multiplex PCR assays. The PCR detection limit was 40, 40 and 400 pg DNA per 25-µL reaction mixture for B. fabae, B. fabiopsis and B. cinerea, respectively. Presence of the broad bean plant DNA in the PCR reactions had no detectable 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. Availability of these species-specific primers will facilitate monitoring the epidemics of chocolate spot of broad bean in the field for implementing appropriate control measures.

Technical Abstract: Botrytis cinerea, B. fabae and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop PCR-based assays to detect and differentiate this three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, 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 (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, 40 and 400 pg DNA per 25-µL reaction mixture for B. fabae, B. fabiopsis and B. cinerea, respectively. Presence of the broad bean 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 monitor the epidemics of chocolate spot of broad bean in the field.