| Pine wilt disease(PWD) is a most devastating disease of pines, so breeding of disease resistance to pine wood nematode(PWN), Bursaphelenchus xylophilus in Pinus massoniana takes an important role in forestry construction. The high-rosin-yield P. massoniana from Xinyi county have been planted in a large area, but the resistance evaluation of Xinyi P. massoniana to PWN has not been studied in guangdong province yet. Therefore, it is necessary to evaluate PWN-resistance of P. massoniana. Since nematodes of B. xylophilus needs to be separated from a large number of samples with inactivation nematodes in the process of resistant evaluation, while the traditional nematode extraction methods are only applicable to the wood samples with live nematodes, therefore a new method for extracting inactivation nematodes from pine wood samples needs to be established. The innovative results have been obtained as follows:1. Descripting the PWD symptoms of three-year-old P. massoniana seedling:(1) PWD symptoms spread from lateral branch, telome or main branch of P. massoniana to the whole plant;(2) PWD symptoms only appeared at the top of the plant, lateral or main branches with yellow or brown needles;(3) Whole seedling died gradually with faded green needles.2. The method of resistance evaluation had been optimized forthree-year-old P. massoniana seeding's resistance to PWD. After inoculated for 122 days, the resistance of the P. massoniana had been evaluated as five grades including grade zero, highly resistant; grade one, moderately resistant; grade two, lowly resistant; grade three, susceptible; grade four, highly susceptible. There was significant PWD symptom difference among each grade. Grade one, high resistance of seedlings had no symptoms; Grade two, moderate resistance started to get sick in the later stage(inoculated for 118 days), their needles turned yellow under one-fourth; Grade three, low resistance started to get sick in the middle and later periods(inoculated for 100 days), their needles turned yellow between two-quarter to three-quarter or turn red under quarter; Grade four, susceptible P. massoniana seedling got sick in early stage(inoculated for eight to twenty days), three-quarters of needles turned red, and it died between five to thirty days. At the same time, the average number of nematodes was checked at each resistance level. The number of nematodes increased gradually with their symptom development, however, when the plants were completely dead, the number of nematodes decreased due to the lack of water.3. There existed significant resistance level difference among three-year-old P. massoniana seedlings. In this study, we inoculated thirteen three-year-old P. massoniana seedlings with B. xylophilus for the first times and another six three-year-old P. massoniana seedlings with B. xylophilus for the second times. The thirteen seedling's resistance level to PWD were evaluated as 5 seedlings at grade zero, highly resistant; 1 seedling at grade one, moderately resistant; 2 seedling at grade two, lowly resistant; 2 seedling at grade three, susceptible; 3 seedling at grade four, highly susceptible, respectively. While that of six seedlings were evaluated as 4 seedlings at grade zero, highly resistant; 1 seedlings at grade one, moderately resistant; and 1 seedlings at grade four, highly susceptible, respectively. The morbidity and mortality of thirteen three-year-old P. massoniana seedlings were 61.54% and 30.77% respectively, but those of six three-year-old P. massoniana seedlings were 33.33% and 16.67% respectively. Therefore, it is necessary to do seedling resistance screening to PWD before planted.4. A new isolation technique ofinactivation nematodes had been established. Inactivation nematodes had been isolated successfully with complete morphological characteristics.They could be used for the count of nematodes and PCR identification.5. This study had also optimized some parameters related to technique of separating inactivation nematodes. The optimal enzyme concentrations [Cellulase/pectinase(mg/?L)] were determined as 0.005/0.001mg/?L, 0.01/0.002mg/?L and 0.005/0.001mg/?Lat frozen, normal and low temperature environment. Meanwhile, the best time for enzymolysis was 6h.6. There are linear correlations between the number of nematodes that separated by Behrman-Funnel and that by enzymatic hydrolysis. Using Microsoft Excel for unitary linear recursiveanalysis of these two sets of data, the regression equations was Y=1.1748X+10.226, R2=0.9074(at frozentemperature); Y=1.2687X-66.6, R2=0.86056(at roomtemperature) and Y=1.1606X-69.2, R2=0.89228(at low temperature) respectively, and R2 fell between 0 and 1. Thus, we can come to the conclusion that the number of nematodes separated by Behrman-Funnel and enzymatichydrolysis method has a good positive correlation. |
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