A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the J...A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.展开更多
Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in ho...Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in hosts that differ in resistance,we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction.Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment,and changes in PWNs after inoculation were assessed.q RT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification.The results showed that the migration and reproductive abilities of PWNs in P.thunbergii were stronger than those of PWNs in P.taeda.After 7 d,the number of nematodes in P.thunbergii was approximately 3.2-fold higher than in P.taeda.After 15 d,the number of nematodes in P.thunbergii was approximately twofold higher than that in P.taeda.Because PWN can adjust its sex ratio after infection,we compared the sex ratio of uninoculated PWNs,to that in the two pine species.In P.thunbergii,the female to male ratio first decreased and then stabilized over time;in P.taeda first decreased and then increased.Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body;the accumulation rate in P.thunbergii was higher than in P.taeda at 7 d,but lower after 15 d.Scanning electron microscopy(SEM)showed significantly more bacteria on the surface of PWNs in P.taeda compared with PWNs in susceptible P.thunbergii.At 12 h after inoculation,the expression of genes related to cell-wall degradation(Bx Beta1-4 and Bxpel1),effectors(Bx CDP1,Bx Sap B1),and active oxygen metabolism(Bxy-ctl-1 and Bx GST3)was 2–6×higher in the resistant pine than in the susceptible one.In contrast,in PWNs,the expression of autophagy-related genes Bx ATG1 and Bx ATG16 was 1.5–2 times higher in P.thunbergii than in P.taeda.These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence.This study will help elucidate the adaptive mechanisms of PWN in different pine trees.展开更多
44 isolates of fungi were isolated from the xylem of the Pinus thunbergii samples(healthy stem,weak stem and withered stem)infected by the pine wilt disease caused by pine wood nematode,Bursaphelenchus xylophilus,from...44 isolates of fungi were isolated from the xylem of the Pinus thunbergii samples(healthy stem,weak stem and withered stem)infected by the pine wilt disease caused by pine wood nematode,Bursaphelenchus xylophilus,from infected trees at Zhoushan and Pinghu of Zhejiang Province in China,and these isolates were identified as:Trichoderma spp.,Penicillium spp.,Pestalotia spp.These identified isolates took 70.62% of total isolates of the fungi.Among the 12 isolates unidentified,one sporiferous fungus was found,the other 11 isolates were nonsporiferous.Trichoderma spp.was the dominant fungus in P.thunbergii,the total rate was 53.34%,followed by Penicillium spp.,18.89%.展开更多
文摘A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.
基金supported by the major emergency project in science and technology of National Forestry and Grassland Administration(ZD202001)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in hosts that differ in resistance,we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction.Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment,and changes in PWNs after inoculation were assessed.q RT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification.The results showed that the migration and reproductive abilities of PWNs in P.thunbergii were stronger than those of PWNs in P.taeda.After 7 d,the number of nematodes in P.thunbergii was approximately 3.2-fold higher than in P.taeda.After 15 d,the number of nematodes in P.thunbergii was approximately twofold higher than that in P.taeda.Because PWN can adjust its sex ratio after infection,we compared the sex ratio of uninoculated PWNs,to that in the two pine species.In P.thunbergii,the female to male ratio first decreased and then stabilized over time;in P.taeda first decreased and then increased.Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body;the accumulation rate in P.thunbergii was higher than in P.taeda at 7 d,but lower after 15 d.Scanning electron microscopy(SEM)showed significantly more bacteria on the surface of PWNs in P.taeda compared with PWNs in susceptible P.thunbergii.At 12 h after inoculation,the expression of genes related to cell-wall degradation(Bx Beta1-4 and Bxpel1),effectors(Bx CDP1,Bx Sap B1),and active oxygen metabolism(Bxy-ctl-1 and Bx GST3)was 2–6×higher in the resistant pine than in the susceptible one.In contrast,in PWNs,the expression of autophagy-related genes Bx ATG1 and Bx ATG16 was 1.5–2 times higher in P.thunbergii than in P.taeda.These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence.This study will help elucidate the adaptive mechanisms of PWN in different pine trees.
文摘44 isolates of fungi were isolated from the xylem of the Pinus thunbergii samples(healthy stem,weak stem and withered stem)infected by the pine wilt disease caused by pine wood nematode,Bursaphelenchus xylophilus,from infected trees at Zhoushan and Pinghu of Zhejiang Province in China,and these isolates were identified as:Trichoderma spp.,Penicillium spp.,Pestalotia spp.These identified isolates took 70.62% of total isolates of the fungi.Among the 12 isolates unidentified,one sporiferous fungus was found,the other 11 isolates were nonsporiferous.Trichoderma spp.was the dominant fungus in P.thunbergii,the total rate was 53.34%,followed by Penicillium spp.,18.89%.
