Location: Crop Bioprotection Research
Project Number: 5010-22410-024-001-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 30, 2021
End Date: Sep 30, 2022
Objective:
Determine if phoretic mites can inoculate ambrosia beetle galleries with beneficial microbes (antagonistic to the fungi or entomopathogenic).
Approach:
Phoretic mite species reared on the selected beneficial microbial species will be evaluated in terms of their ability to carry and inoculate microbial propagules into ambrosia beetles’ galleries. Thirty female mite individuals will be either manually isolated from the cultures being maintained on fungi or isolated from the regular stock colonies and dusted with spores of beneficial fungi. The number of spores/propagules that can be loaded on to a mite will be determined by allowing mites to walk on avocado bolts for different time periods after which microbe DNA will be extracted and quantified using qPCR. Females carrying beneficial microbes will be transferred to ambrosia beetle colonies being kept in rearing tubes. Each microbial species will be considered as one treatment (n = 5). Another treatment will consist of surface sterilized female mites in 70% EtOH for 30 seconds. For the control replicates, no mites will be added to the rearing tubes. The transferred mites will be given enough time to reproduce and inoculate the galleries. After 30 days, the galleries from rearing tubes will be dissected under a stereo microscope and the number of mites and ambrosia beetles will be recorded. Infection by entomopathogenic fungi will be assessed in ambrosia beetle individuals. DNA and microbial isolates will be recovered from the galleries in order to verify the impact of the mites on the microbial ecology of the gallery. Parts of galleries will be shipped to the ARS for beneficial microbe analysis. The microbiome of the gallery will be evaluated based on standard 16S and ITS amplicon based next generation sequencing methods.
A similar trial will be conducted in the field. Avocado logs will be placed in the field for two weeks next to laurel wilt infected trees. During this period, it is expected that ambrosia beetles will infest and bore galleries into the clean logs. Then, 200 phoretic mite females, will be either manually isolated from the cultures being maintained on fungi or isolated from the regular stock colonies and dusted with microbial propagules. The females will be placed on the logs and left in the dark at 26 ± 1°C, 70 ± 10% R.H. for two days. Thereafter, the logs will be placed back in the field. Each microbial species will be considered as one treatment (n = 5). Another treatment will consist of surface sterilized female mites in 70% EtOH for 30 seconds. For the control replicates, no mites will be added to the logs. After 30 days, the galleries will be excavated and evaluation of the trials will be conducted as described for the previous experiment.
