| Pine Wilt Disease(PWD)has caused severse losses to pine trees producting worldwide,Pinus massoniana which is the key widely species for construction of ecological civilization in China.The unique and important economic value of P.massoniana makes it play an irreplaceable role in the process of poverty alleviation and rural rejuvenation in China.However,in recent years,PWD has caused heavy loss in the P.massoniana ecological forests in 17 provinces across China.At present,the cost of preventing and controlling PWD is expensive.The main reason is that the pathogenesis of PWD has not been celuciated,so it is difficult to design its targeted precise prevention and control measures.Highly sensitive P.massoniana is a necessary material to reveal the pathogenesis of PWD.Because the highly sensitive pine dies after inoculated with PWN,and the potential highly sensitive PWD one cound not be identified till today,which makes it impossible to carry out in-depth research on the pathogenesis of P.massoniana.Therefore,it is urgent to establish the rapid PWD screening technology of PWD in vitro.This study revealed the symptom development and PWN number change of highly-sensitive P.massoniana of PWD,and established the identification technology of PWN in vitro.The main innovative achievements are as follows:1.The PWD symptom development highly sensitive seedlings of P.massoniana in different ages were revealed.The artificial inoculation PWN method was carried on semiannual,one-and-a-half-year,two-and-a-half-year,and four-year-old P.massoniana seedlings.It was found that all the highly sensitive plants would die after the onset of the disease,but the symptom development process were different.The PWD symptom on highly sensitive half-year P.massoniana seedlings occures on the 5th day after inoculation,and the symptoms expanded rapidly,the whole plant withered and died within 4 days.The PWD symptom on highly sensitive one and a half year,two and a half years,and four years old P.massoniana seedlings began on the 8th,10 th,and 19 th day after inoculating with PWN,and died 11 days after symptoms.one and two-and-a-half-life highly sensitive P.massoniana seedlings began to die 11 days after symptoms,and four-year highly sensitive P.massoniana seedlings began to die 17 days after symptoms.The symptoms of one-and-a-half-year,two-and-a-half-year,and four-year-old highly susceptible seedlings of P.massoniana are as follows: firstly,the needles at the inoculation site of the shoots loses water,loses green and yellow,slightly drooping and curling,and some droop at the top;Then,the needles of the seedlings of P.massoniana begin to lose green and yellow from the top to the outside,and expand downward at the same time,and the needles of 1/4～1/2 and 1/4 of the seedlings turn red;with time going on,the needles of 1/2～3/4 of the seedlings of P.massoniana drooping,yellowing and reddening;at the end,more than 3/4 of the needles turned yellow and reddening,and the whole plant withered and died.2.The PWN number change in high-sensitivity P.massoniana seedlings were revealed.Under the same conditions,one and a half year old P.massoniana seedlings were inoculated with PWN.It was found that the number of PWN in different resistance levels of P.massoniana seedlings had significant differences in the initial number of invasion,the propagation rate of PWN after artificial inoculation 9 days keeps low,the population density of PWN was lower in different resistant level of P.massoniana seedlings.Then,The PWN number in the highly susceptible seedlings of P.massoniana began to increase significantly.After 17 days of inoculation,the number of PWN increased significantly in a sudden manner,reaching the maximum value at 21 days,and the average increment rate was 42.67pieces/g/day.The PWN number in mid-sensitive P.massoniana seedlings increased significantly after 13 days of inoculation,and reached the maximum value at 21 days of inoculation.The average increment rate was 1.68-12.34/g/day.However,the number of PWN in the seedlings with high resistance to P.massoniana was very few,and the average increment rate was 0.097/g/day.The results lay a foundation for the establishment of rapid identification technology of highly susceptible PWN P.massoniana in vitro.3.Established a new rapid identification technology for highly susceptible PWN of P.massoniana in vitro.The specific operations are as follows: for every 10 P.massoniana seedlings as a group,cut the top of the new shoot of the current year’s P.massoniana,pull out its the needles and leaves of the isolated shoot of P.massoniana,soak in the suspension of PWN with 5000,and treat them at 25℃ for 24 hours,the residual nematodes in the epidermis of the branches were washed with distilled water,the number of initial invading nematodes was separated,and their significant differences were analyzed by significant analysis.The group tree with the highest PWN number was identified as highly susceptible P.massoniana with to PWN. |
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