Isolation,Identification And Virulence Of The Entomopathogenic Fungus BXY01 Strain Of Atrijuglans Hetaohei

Atrijuglans hetaohei Yang?Lepidoptera:Heliodinidae?is a pest specially in walnut fruit.The damage of A.hetaohei is by larvae boring into walnut fruit to eat the pericarp and seed.After damaged,pericarp and fruit become black and shrinks,that caused a great economic loss.In current,the primary measure for controlling A.hetaohei is to spray chemical pesticides to kill imagoes,eggs,and new hatchling larvae,but it is ineffective for the larvae that have bored into the walnut fruit.In addition,chemical pesticides can lead to environmental pollution and residue problems.Based on the habit of the mature larvae of A.hetaohei overwintering in soil for 9 months,we study the entomogenous fungus as biological control agent in the soil to infect A.hetaohei larvae.The contents:1.In the experiment orchard in Yuxian,Shanxi.Insect population was surveyed,and the development progress of larvae in walnut fruit and damage of the fruit were investigated.The chemical compositions protein,fat,chitin,amino acid in mummificaiton of larvae were analyzed,so as to provide basis for biological control.2.A strain was isolated from the natural cadaver of A.hetaohei.Inoculation of the strain on the host insect was conducted.The strain was identified based on morphological and molecular.3.The virulence of BXY01 strain and the other 3 strains of Beauveria bassiana to A.hetaohei larvae was tested and compared.4.The biological characteristics and persistence of the strain in soil was tested.The results:1.The insect population,damage of the fruit,chemical composition1)The density of insect population,characteristic of larvaeInvestigation shows that in March and April,the density of insect population is 78^-100 m-2,including living cocoons 9^-18 m-2.Mature larvae in cocoons are 5-7mm in length with tawny head and white body.Naturally infected larvae are 1^-1.5 m-2.2)Development progress and damage of larvae in walnut fruitBy dissecting and observing the damaged walnut,it was discovered that at the preliminary stage,pericarp of the walnut is still green,the galleries and holes caused by larvae are tiny;at medium-term,pericarp of the walnut develops some dark spots,the larvae have grown up,the fruit flesh have been eaten,galleries in the walnut fill with insect feces;At later stage,most pericarp of the damaged walnut turns black,most of the flesh have been eaten,there are plenty galleries and holes that were filled with insect feces;At final stage,larvae are maturity,pericarp of the damaged walnut have completely turned black,the flesh have been eaten,only insect feces were left.The average number of the larvae in one walnut fruit is 6-7,even reaches 13.Mature larvae come out of fruit,and drill into soil.The mature larvae are 6-9mmin length with tawny head and light red body except last two segments of abdomen in white.3)Chemical compositions of larval mummificationThe contents of protein,fat,chitin in the larval mummification are 51g/100g,28g/100g,and 21g/100g,respectively.The contents of amino acid are 47.24g/100g,in which,Gln and Asp acid with the highest contents are 6.02g and 5.17g,respectively.2.Isolation,purification and identification of entomopathogenic fungus BXY01 strain from A.hetaoheiNatural cadavers of A.hetaohei larvae infected by fungus were found in the overwintering cocoons.Their surface covered with white hyphae.From which,a strain BXY01 was isolated and proved as a pathogen of A.hetaohei by reinoculation test on host insect.The colonies of the strain were with dense hyphae and regular edge.The colonies viewed by back were white in early cultivation and yellow in later stage.Hyphae were white,and the conidiogenous cell extended at upper into a "Z"-bending conidiogenous axis.Based on the morphological characteristics identification the strain was identified to be Beauveria bassiana.By sequencing and analyzing rDNA ITS region of the strain BXY01,and submiting to GenBank?Accession No KU214583?,the sequence was aligned with other sequences in the GenBank.The result showed that the strain BXY01 was homology in 99%with Beauveria bassiana?Accession No.JN379794?No.JN379807?No.JN379808?from GenBank.Thus,the molecular evidence pointed the strain BXY01 to be Beauveria bassiana.3.The virulence of BXY01 strain and the other 3 strains of Beauveria bassiana to A.hetaohei larvaeOther 3 strains of Beauveria bassiana,TST05?from Carposina niponensis Walsingham?,FDB01?from Acantholyda parki?,SYN01?from Dendrolimus kikuchii?were compared with BXY01,by inoculation with 10⁸ mL^-1 spore suspension to A.hetaohei larvae.The adjusted mortality rates of the larvae were 84.4±4.5%,87.8±5.2%,91.1±2.4%,and 93.3±4.3%,respectively,at 8th day after treatment.LT50 were 4.743d,4.934d,4.098d and 3.760d.It indicated that the four strains are high virulence to A.hetaohei.The strain BXY01 is the highest virulence,to A.hetaohei.By inoculation with spore suspension of BXY01 in concentration 1×10⁹ mL^-1,5×10⁸ mL^-1,1×10⁸ mL^-1,5×10⁷ mL^-1 and 1×10⁷mL^-1,the adjusted mortality rates of the larvae were 100%,95.56±4.2%,93.3±4.3%,85.7±12.3%,and 76.7±7.2%,respectively,at 8th day after treatment.It indicated that a larger concentration of BXY01 spore suspension leads to a higher adjusted mortality rate and a shorter LT50.4.Biological characters and the persistence of BXY011)Biological characters:The BXY01 strain can adapt to a wider temperature range.The mycelia growth,conidia yield and conidia germination can occur in 15-35?.25?is the best temperature for the strain,in which,the strain is with the largest colony diameter?3.87±0.03cm?,the most conidia yield(11.48±0.81×10⁷mL^-1),and the highest conidia germination rate?67.5±4.5%?.After induced by UV rays,the strain is reduced in colony diameter,spore production,spore germination rate,but it has no significant change.It proved that the strain has the anti-ultraviolet ability.2)The persistence of BXY01 strain in field soilThe fungal powder was prepared and then scattered into soil with the depth from 0^-15cm in the orchard on September.The survived population of the strain in soil depth 0cm,5cm,10cm,and 15cm were determined month by month.On September,the CFU?colony forming unit?were 102.38±6.29×10⁴g^-1.On October,the CFU were 27.76±1.89×10⁴,42.04±3.21×10⁴,35.44±2.16×10⁴,21.97±2.13×10⁴ g^-1.On November,the CFU were 8.35±1.35×10⁴,19.10±1.53×10⁴,14.31±1.80×10⁴,11.11±1.83×10⁴g^-1.On December,the CFU were 1.22±0.25×10⁴,8.27±1.01×10⁴,5.89±0.93×10⁴,3.50±0.57×10⁴g^-1.On February,the CFU were 0.42±0.16×10⁴,4.36±0.33×10⁴,4.75±0.57×10⁴,3.74±0.34×10⁴ g^-1.On March,the CFU were 0.1±0.07×10⁴,4.37±0.36×10⁴,4.63±0.51×10⁴,3.72±0.58×10⁴g^-1.It indicated that survived population of the strain declined obviously from September to November,but it waved in certain range from December to March.The survived population was higher in depth of 5^-10cm than the 0cm and 15cm.The strain possesses a higher survive ability in the field condition and was propitious to sustainable control of A.hetaohei.