Biological characteristics of Bacillus species in the suppression of Bipolaris cactivora causing stem rot of grafted cactus

Abstract

Grafted cactus composed of two cactus species Hylocereus trigonus and Gymnocalycium mihanovichii as stock and scion, respectively, is an ornamental plant, for which its major production area is Korea that comprises about 70% in the world market. One of the most important limiting factors for the production of grafted cacti in Korea is the qualitative and quantitative yield loss derived from stem rots of the cactus especially caused by Bipolaris cactivora. Control measures of this disease have not been well established until now. Thus, the purpose of this study is to develop microbial control agents useful for the control of the Bipolaris stem rot in the grafted cactus. Two antifungal bacterial isolates (GA1-23 and GA4-4) out of 943 microbial isolates finally selected for the biocontrol of the stem rot had cream-colored, flat or raised, and undulated colonies, rod shaped bacilli, Gram-positive, which were identified as Bacillus subtilis and B. amyloliquefaciens, respectively, by the cultural characteristics and Biolog program and 16S rRNA sequencing analyses. These two bacterial isolates showed both 40% inhibition rates of conidial germination on agar (solid) culture conditions, and 30% and 50% inhibition rates, respectively in broth (liquid) conditions. Also, they inhibited over 50% for the mycelial growth of other cactus pathogens such as Alternaria, Botrytis, Colletotrichum, Fusarium species. In vitro assay for the control of the stem rot using cactus stem discs revealed that the control efficacy of the bacterial isolates was higher in their pre- treatment than simultaneous and post- treatments. In vivo test for the biocontrol of the stem rot showed that only simultaneous treatments of the bacterial isolates reduced the disease severity significantly (control value of 64.3%), while no significant disease reduction of disease severity occurred by their pre- and post- treatments, compared to the control. To know the mechanism of their antifungal activity, fungal hyphae and spores were observed by light and scanning electron microscopy. Light microscopy of the pathogen treated with the bacterial isolates showed the inhibition of spore germination with malformation of germ tubes. Ten days after inoculation, conidia inoculated with antifungal bacteria formed circle-like vesicles. Scanning electron microscopy of the pathogen treated with the bacterial isolates showed the inhibition of spore germination with malformation of germ tubes and hyphal disruption sometimes accompanied by hyphal swellings adjacent to the bacteria. All of these results suggest the two bacterial isolates have a good potential to be developed as biocontrol agents for the Bipolaris stem rot of grafted cactus.