Exploration Of Novel Prophylactic From Aromatic Plants Against The Asian Citrus Psyllid

The Asian citrus psyllid（ACP）,Diaphorina citri Kuwayama（Hemiptera:Psyllidae）,is a phloem-feeding insect native to Asia,it is the vector of（HLB）Huanglongbing（citrus greening）,caused by a phloem-limited bacterium?Candidatus Liberibacter asiaticus（Ca.L）.The Huanglongbing（citrus greening）is considered one of the devastating pests of citrus worldwide.The natural transmission of HLB is by Asian citrus psyllid.Huanglongbing was identified from the phloem tubes of the affected citrus plant leaves,HLB species are the obligate parasites of the plants and only able to multiply in the body of their eukaryotic hosts especially insects.Currently,there is no cure for HLB has been reported.So the main reliance is to control the vector ACP.The primary control of ACP mainly depends on the use of broad-spectrum synthetic insecticides,which negatively affects the environment,humans,natural enemies and development of resistance.Recently,resistance against some renowned classes of insecticides,including pyrethroids,organophosphates,neonicotinoids,and carbamates has been reported in citrus psyllid.Therefore,there is a need for novel management strategies,such as attractants,repellents,antifeedants,and toxicants from plant sources,especially from non-host plants,which will possibly serve as an alternative to synthetic insecticides.Botanical pesticides are the potential alternatives to synthetic chemical pesticides.Due to their complex and multiple modes of actions showed its advantages in reducing the economic stress and pest resistances from synthesized chemicals pesticides.Here we evaluate the behavioral and toxic effect of Artemisia absinthium essential oil and Sophora alopecuroides extract against ACP.Furthermore,the selected plants chemical analysis were done by using GC-MS.The results will have important implications on the novel management of D.citri.The main results are the following:1.The chemical composition of the essential oilThe common wormwood Artemisia absinthium（L.）is an aromatic perennial herb,which is wildly grown in the Skardu Baltistan.Locally its fresh and dried form is used as both medicinal and insecticidal purposes.This study first time reports the chemical composition of hydro-distilled essential oil obtained from aerial parts of A.absinthium.Upon hydro-distillation,it gave dark green colored oil with a yield 0.41%（w/w）（based on sheltered dried plant parts）.The variation in chemical composition and their relative percentage in the essential oil of A.absinthium are cited to vary qualitatively and quantitatively according to geographical location and environmental conditions.The GC-MS analysis of A.absinthium essential oil obtained from the population collected from Skardu Baltistan also showed variation in chemical composition and their relative percentage.The oil was dominated by sesquiterpenes（61.01%）and monoterpenes（17.92%）.The major dominating constitutes were guaiol（19.33%）,α-bisabolol（8.83%）,carveol（6.16%）,chamazulene（5.94%）,limonen-6-ol,pivalate（5.37%）and geranyl-α-terpinene（5.63%）,which accounts overall 51.62%of total constitutes identified.2.Toxicity and enzyme inhibition activities of the essential oil and its dominantconstituents against adult Asian citrus psyllidsThe extensive use of synthetic chemical insecticides has led to negative effects on the environment and human health.Therefore,scientists are in search of natural products,especially those from plant sources,which are less hazardous,eco-friendly,and effective alternatives to conventional pesticides.Essential oils are potential alternatives to synthetic chemical insecticides.However,the toxicity of Artemisia absinthium against Asian citrus psyllid D.citri has not been documented.Therefore,here we focused on the A.absinthium essential oil（ABEO）extracted from the population grown under the climatic conditions of Pakistan‘s high plateaus,as a natural pesticide for the control of the citrus psyllid.The results indicated that the oil caused significant acute mortalities in both topical and residual contact applications,with 50%lethal dose(LD₅₀)and 50%lethal concentration(LC₅₀)values of 5.2μg/insect and 24.4 mg/m L.Among the constituents tested only carvacrol,（-）-α-bisabolol and chamazulene were toxic,with LD₅₀ values of 6.9,8.2,and 10.3μg/insect via the topical application and LC₅₀ values of 28.5,33.5,and 39.4mg/m L via residual contact exposure.Several combinations of dominant constituents were both additive and synergistic when applied topically,especially carvacrol+（-）-α-bisabolol+chamazulene（1:1:1）displayed synergistic effect,with a poison ratio of 1.39.Furthermore,the essential oil and carvacrol inhibit acetylcholinesterase activity in citrus psyllid.It has been concluded that the essential oil and some of the compounds from A.absinthium might be developed as eco-friendly approaches for the control of citrus psyllid.3.Interference mechanism of alkaloids extract on host finding and selection of the Asian citrus psyllidManipulating insect behavior through the deployment of semiochemicals offers a promising opportunity for protecting crops in a sustainable manner.Therefore,there is still a significant opportunity for the development of natural crop protectants as eco-friendly tools in pest management.In this context,the aim of the current investigation is to find a novel prophylactic against the Asian citrus psyllid（ACP）and to gain a better understanding of the host-finding and selection ability of the ACP towards Murraya paniculata seedlings treated with Sophora alopecuroides alkaloids extract（SAAE）.Our results indicate that foliar application of SAAE influences the psyllid host-finding and selection process.The behavioral assay with M.paniculata seedlings treated with 15 and 30 mg/m L of SAAE,with masked visual cues,revealed that only 6.6 and 10.4%psyllids were able to locate the host in the vials.The results also indicate that citrus psyllids mainly rely on both visual and olfaction in host-finding and selection.In choice settling experiments,psyllids settled almost completely on control seedlings rather than on seedlings treated with SAAE at a concentration of 30 mg/m L.Chemical analyses of the alkaloids extract revealed the presence of sophocarpine（33.90%）,sophoridine（6.23%）,anagyrine（2.77%）,matrine（2.38%）,lupanine（1.68%）aphylline（0.89%）,and sophoramine（0.75%）.In further behavioral bioassays with the dominant alkaloids sophocarpine and sophoridine,the alkaloids repelled ACP at higher concentrations of 50 and 70 mg/m L as compared to SAAE.Furthermore,the 50 mg/m L（1:1,v/v）combination of sophocarpine and sophoridine displayed a synergistic effect and showed the maximum behavioral effect as compared to the individual alkaloid.Based on our results,SAAE makes M.paniculata seedlings unattractive to the psyllids,and therefore,alkaloids could be used in reducing the colonization of citrus plants,subsequently curtailing HLB infection.4.Development and evaluation of emulsifiable concentrate formulations for novel management of the Asian citrus psyllidUtilization of non-host plants semiochemicals to mediate the insect behavior offers a promising opportunity for novel management of insect pests in field crops and fruits.Therefore,there is still a substantial opportunity for the development of natural prophylactic as an eco-friendly approach in novel pest management programs.Sophora alopecuroides extract has been used as a natural pesticide in the control of agricultural and household pests,but the low persistence effect and rapid biodegradability limit its use on a wider scale in pest management programs.In this study,an emulsifiable concentrate formulation containing S.alopecuroides extract（SAE-EC）was developed with a simple procedure and evaluated for its ovicidal,antifeedant and repellent effects against Diaphorina citri under laboratory and semi-field conditions.Our results indicated that SAE-EC at 15,30 and 50 mg/m L concentrations provides complete protection against psyllids for a period of 96 h after application both under semi-field conditions,while the aqueous methanolic extract of S.alopecuroides loses its persistence 48 h after application.Furthermore,the emulsifiable concentrate at 20 and 30 mg/m L concentrations,only 15.97%and 31.97%of eggs were able to hatch,and at similar concentrations,72.86%and 85.5%of honeydew secretion were reduced as compared to the control.SAE-EC at 30 mg/m L concentrations has not shown any phytotoxic symptoms on Murraya paniculata seedlings.Fourier transform infrared（FTIR）study revealed the presence of alkaloids in emulsifiable concentrate after three months of its preparation placed under ambient temperature.Furthermore,the particle size and polydispersity index（PDI）of the emulsifiable concentrate were also confirmed by dynamic light scattering（DLS）.Our finding indicated that emulsifiable concentrate formulation prolongs the persistence of S.alopecuroides extract and enhances its efficacy under both laboratory and semi-field conditions.It has been concluded that the emulsifiable concentrate formulation containing S.alopecuroides extract might be developed as an eco-friendly novel prophylactic against citrus psyllid.