Standardization Of Technology For Evaluating Plant Resistance To Pine Wilt Disease Of Pinus Massoniana Seedlings Caused By Bursaphelenchus Xylophilus

Pine Wilt Disease(PWD)is caused by Bursaphelenchus xylophilus.The pathology of PWD is complicated and difficult to control.Resistance breeding has become an important approach to defense against PWD.PWD has occurred in 17 provinces across China.Some provinces such as Anhui and Jiangsu have already selected PWD resistant varieties suitable for local cultivation and have promoted planting in large areas.Till today there is no similar report on controlling PWD in Guangdong Province.Therefore,there is an urgent need to conduct selecting PWD resistant pine seedling.In this study,the seedlings of Tongmian Pine from the Guangxi Zhuang Autonomous Region were used to carry out the research on seedling evaluation technical standardization.The mainly innovative results have been obtained as follows:1.A three-month seedling epidermal scratch inoculation technique was established.The specific operation is as follows: two-1.5cm-long wounds were made with a sharp knife along stem 3cm below pine seedlings shoot where needle leaves were pull down.A small funnel was made surrounding the lower end of two-1.5cm-long wounds with parafilm to keep nematode suspension which was injected for inoculating pine wood nematode(PWN)into pine seedlings.Then the whole seedlings were put in a plastic bag for two days to keep humid.PWD symptom occurred on seedlings 7-10 days after inoculation.Seedlings died after 18 days.And the three-month seedling epidermal scratch shoot inoculation of young shoots was verified on P.thunbergii Parl..2.The inoculation PWN number suitable for evaluating the six months pine seedling was selected.Six months seedlings of P.massoniana were inoculated with 200,500,1000,2000,3000,4000 and 5000 PWN respectively.200 PWN did not led to any PWD symptoms on pine seedlings.PWD symptoms occurred after 7 days inoculation with 2000 or 3000 or 4000 or 5000 PWN,while it occurred after 13 days inoculation with 500 or 1000 PWN.Until 21 days inoculation,there is no significant difference on plant death rate inoculated with 3000 or 4000 or 5000 PWN.Therefore,3000 PWN are the optimal PWN inoculum number for a half-year-old pine seedling.3.PWD symptom developing process were described for P.massoniana seedlings with different resistance level.No PWD symptom occurred on highly resistant seedlings of P.massoniana.For resistant seedlings,PWD symptom developed slowly 8 days after inoculation.After 10 days of inoculation,the needle leaves around the inoculation site of Tongmian Pinus seedlings were yellowed and the needles were drooping on the shoots;The inner 1/2 needles was wilt,chlorotic,curled and drooping.After 16 days of inoculation,3/4needle leaves wilt,chlorotic,and curl,leaving only a small amount of external needle leaves green.After 19 days,the whole plant yellowed and died.While PWD symptom developed faster on sensitive pine seedlings 8 days after inoculation,the whole plant died13 days after inoculation.4.The trends of population dynamics of PWN amplification in pine seedlings were found.The trends were not the same,depending on whether they were highly resistant plants or highly sensitive plants.The high resistant plants had a quick descending trend,and the high sensitive plants had a slower downward trend.The high resistant plants had the fastest decline at 12-24 hours and were basically stable at 36-96 hours;After 4 days,the number of PWN in the pine woods decreased significantly.The number of PWN invaded into high sensitive plants was higher and basically remained at the stable stage from 24-60 hours and 72-120 hours;on the 6th day,the numbers of PWN began to rise,indicating that the nematodes have successfully bred.The study on the change trend of the number of PWN in pine seedlings at different inoculation time lays a foundation for further revealing its resistance mechanism.5.The culture medium for inducing callus of Xinyi P.massoniana were studided.The suitable medium for inducting callus from spring shoot tip of P.massoniana is that contains DCR+0.5 mg/L NAA+2 mg/L 2,4-D+1.5 mg/L 6-BA+0.2 g/L Vc+500 mg/L CH+450 mg/L Gln.The medium with 3g/L myo-inositol was used as the subculture medium;the callus was transformed from dark culture to light culture containing different concentrations of TDZ,and added 0.6mg/L TDZ which is the most suitable concentration.The green callusnumber induced in the base was the most,and the brown callus number was the least.This result will lay the foundation for the preservation and propagation of resistant P.massoniana materials.