Endophytic Colonization Of Entomopathogenic Fungi In Citrus Limon For The Control Of Diaphorina Citri(Hemiptera: Liviidae)

The ability of entomopathogenic fungal strains of the genera Beauveria,Isaria and Metarhizium to colonize Citrus limon plants and the effects of their systemic colonization on plant growth,survival,development and fecundity of Diaphorina citri was examined in this study.Preliminary fungi pathogenicity studies were conducted to ascertain the degree of virulence of the selected fungal strains against adults of Asian citrus psyllid(ACP).Examined fungal pathogens induced over 60% ACP adult mortality within 5 days of exposure,while,two strains of Beauveria bassiana caused 92-95% mortality of adult ACP after 7 days of exposure.The underlined fungal entomopathogens colonized various parts of the plant,leaf,stem and root tissues following foliar treatment of citrus plants with their fungal conidia suspension,while two strains of B.bassiana were sustained in colonized plants up to 12 weeks.The inoculation methods used(foliar spraying and soil drenching)and the age of citrus seedlings at the time of inoculation(3,4 and 6 months-old)were found to significantly influence endophytic colonization success.This was evident in the higher colonization percentage that was recorded in the foliar treated seedlings(up to 58.3%)in comparison to the soil drenched seedlings(up to 25%)and higher fungal reisolation rate that was recorded in the younger seedlings respectively.The height and leaf number of colonized seedlings were generally improved by endophytic B.bassiana.In the experiment conducted to examine the effects of systemic endophytic colonization on survival,development and fecundity of D.citri,endophytic B.bassiana was found to mediate increase in adult mortality,reduction in fecundity and emergence rate of ACP nymphs and adults.Endophytic B.bassiana induced 15% adult mortality and reduction in nymph and adult emergence across three successive ACP generations.Generally,female ACP feeding on endophyte-challenged seedlings laid fewer number of eggs in comparison to those feeding on endophyte-free seedlings.In addition,entomopathogenic fungal strains naturally affecting D.citri on the field were isolated,characterized and identified using morphological observation,supplemented by analysis of the ITS sequence data.The new isolates were confirmed to belong to the genus Isaria following the analysis of their phylogenetic relationship with other fungal isolates through Maximum Parsimony and Maximum Likelihood methods.The new strains were tested in a bioassay test against immature and mature stages of ACP and found to induce similar symptoms to what was observed on field collected mycosed psyllids.One of the two fungal strains,Isaria javanica(IJ-ACP),was successful in colonizing leaf and stem tissues of C.limon following treatment of seedlings via foliar spraying with fungal conidia suspension.Also,the possibility of achieving higher percentage colonization of different plant parts post treatment with entomopathogenic fungi using dual inoculation methods was evaluated.As expected,dual inoculation methods performed better than the single inoculation methods,as systemic colonization of the entire plant was achieved using seed soaking + foliar spraying method in combination.In overall,the findings of this study indicated that the colonization of insect pathogenic fungi in plants can improve seedling height and leaf number,and protect citrus plants from ACP attack.The study provides insights into application of EPF as fungal endophytes as a novel biological pest management technique against the Asian citrus psyllid.