Genetic Diversity Of Asian Citrus Psyllid Population From China And Pakistan,and The Pathogenesis Of Entomopathogenic Fungi On Psyllid

Asian citrus psyllid(ACP),Diaphorina citri Kuwayama(Hemiptera:Liviidae),one of the important pests of the genus Rutaceae and an insect vector of citrus Huanglongbing(HLB).Analysis of pest genetic diversity from different regions is the basis for pest control research.On the base of genetic diversity of D.citri from China and Pakistan,we analyzed the pathogenicity of two Isaria strain against D.citri which fed on HLB-diseased and healthy plants.This research will open a new sight for controlling D.citri.(1)To find out the genetic relationship and diversity among the populations of ACP and associated Candidatus Liberibacter asiaticus(CLas)from two countries(China and Pakistan),sequence data of three different genes,cytochrome oxidase subunit I(COI),Cu-transporting protein(ATOX1)and 16S rRNA,were used to characterize all populations.For ACP populations,both genes presented a significant strong intermingled relationship among all populations and put all population into a single haplotype(Dcit-2),which proved similarity between Chinese and Pakistani populations.Moreover,for CLas strains,16S gene also presented strong relationship for all sampled populations.All three genes of ACP and CLas populations elucidated more than 95%resemblance to each other.On the other hand,a significant genetic variation was observed by three genes for overall populations,although,country wise variation was non-significant between all collected populations.ATOX1 gene presented higher diversity through Fu’s Fs test(π=0.01081,p<0.003)whereas COI gene gave less diversity under Fu’s Fs and Tajima’s D test(π=0.00512,p<0.000 and 0.05,respectively).Similarly,nucleotide mismatch distribution also had shown enough genetic variation among all ACP populations,under both genes.Our sequence data for both genes proved a similar genetic makeup among all ACP populations from both countries.(2)Then,we investigate the development of ACP on citrus(Citrus sinensis)(healthy and HLB-diseased)and jasmine(Murraya paniculata)plants at various temperatures.Development observations were recorded for egg,nymph and adult stages on all plants and three different temperatures(20,25 and 30℃).Results showed that egg(3.02-3.72d),nymph(first stage to forth stage,7.63-9.3d,fifth stage,5.35-5.65d)and adults(24.46-28.47d)that fed on HLB citrus developed faster than that on others.It indicates that CLas,a HLB disease bacterium,can promote the growth of Asian citrus psyllids by promoting the intake of more sap from the plant phloem,but reducing its survival rate.(3)Two new Isaria strains were collected from citrus orchards of Fuzhou(China),and HLB-diseased plants were verified by running PCR for 16S gene of Candidatus Liberibacter asiaticus(CLas).The mortality observations were recorded for the nymph(fifth instar)and adults on all plants at 25℃.The results showed that the mortality rate of Asian citrus psyllids was the highest on the HLB-infected plants.Ⅰ.javanica caused the fastest mortality of nymphs and adults(32.21± 4.47%and 19.33 ± 4.51%)on HLB-diseased plants with the concentration of 1×108 conidia.mL-1 after 3 d and 7 d,respectively.(4)To characterize various metabolites from crude methanol extract of Ⅰ.fumosorosea,HPLC-QTOF MS technique was used,and all compounds were identified through molecular mass and formulae.Overall,seventeen compounds were identified which were categorized into three groups,i.e.,(1)alkaloids(Isariotins A-C),(2)peptides(Bassianolide,Beauverolides,Beauvericin A,Isaridins and Destruxin E)and(3)polyketide(Tenuipyrone).All these metabolites are known as insecticidal agents against various insect pests.On the other hand,bassianolide was assessed against the nymphs and adults of Asian citrus psyllid(ACP),Diaphorina citri Kuwayama(Hemiptera:Psyllidae),in laboratory conditions on healthy and HLB-diseased plants of Citrus spp.Bassianolide caused more than 70%and 80%mortality of nymphs and adults after two days of application,respectively.After three days of chemical application,all nymphal and adult populations of ACP were killed by bassianolide.However,mortality rate of both populations,nymphs and adults,was higher on HLB-diseased plants as compared to healthy plants.The mortality variation might be due to the emission of various volatile compounds from healthy and HLB-diseased plants,and these volatiles might be play role as a synergetic agent with such fungal metabolites.Therefore,there is a need of an extensive molecular work to characterize the chemical profile of volatiles,and to find out the chemical pathway of certain volatiles after the infestation of insect pests.