Taxonomic Identification And Pathogenicity Of Fusarium Species From Wild Apple Forest In Xinjiang

Malus sieversii(Ledeb.)M.Roem.,the wild ancestor of domesticated apple,is the constructive species of wild fruit forest in Tianshan Mountains.It is a rare and endangered plant,and ranked in grade key protected wildlife in China.Its germplasm resource is an important gene pool for apple breeding and improvement.However,in recent years,the wild apple forest in Xinjiang has declined and died in large areas.Symptoms of decline,including xylem browning on wild apple trees are suggestive of infection by Fusarium species.In this study,we selected two main distribution areas of wild apple forest in China from 2016 to 2018 — Xinyuan County and Gongliu County on the northern slope of Tianshan Mountain in Xinjiang,to investigate the disease caused by Fusarium species.The declined wild apple twig samples of xylem browning were collected to for isolating and purifying strains.Based on phylogenetic analysis,morphological observation,optimum growth temperature measurement and pathogenicity tests,the taxonomic identification and pathogenicity of Fusarium species from wild apple forest in Xinjiang were determined.The results of this study will provide a scientific basis for the prevention and control of Fusarium disease in wild apple forest.The main results are as follows:1.The investigation was carried out in wild apple forest in Xinyuan and Gongliu from 2016 to 2018.A total of 362 fungal strains were isolated from 448 samples of the junction of the diseased and healthy parts of the afflicted twigs that showed xylem browning symptoms.Through morphological observation and phylogenetic analysis,59 isolates of Fusarium species that accounted for 16% of the total number of strains were found.2.Six Fusarium species were identified by observation of morphological features and DNA sequence analysis,which included 24 strains of Fusarium avenaceum(Fr.)Sacc.,13 strains of Fusarium tricinctum(Corda)Sacc.,13 strains of Fusarium solani(Mart.)Sacc.,4 strains of Fusarium proliferatum(Matsush.)Nirenberg,3 strains of Fusarium sporotrichioides Sherb.and 2 strains of Fusarium sp.1.Among them,F.avenaceum was the dominant species;F.sp.1 was found only in Gongliu County,and F.sporotrichioides was found only in Xinyuan County.The optimum growth temperature of five identified Fusarium species was 25-30°C in the dark on PDA medium.3.The indoor and field pathogenicity determination of F.proliferatum(PR),F.sporotrichioides(SP),F.tricinctum(TR),F.solani(SO)and F.avenaceum(AV)by the methods of in vitro apple leaves and twigs inoculationand stems and branches inoculation of living apple trees showed that the size of the lesions were significantly different among the five Fusarium species.In all trials,no symptoms were observed on the leaves,twigs and living apple trees inoculated with Fusarium tricinctum.The largest lesions were formed on the detached leaves of five apple varieties,in vitro twigs of Fuji apple and stems and branches of living apple trees infested with F.sporotrichioides.And F.avenaceum was most infested after inoculation of wild apple twigs in vitro.The order of pathogenicity of the five Fusarium species to the apple twigs in vitro was:(1)wild apple twigs: AV>SP>SO>PR>TR;(2)Fuji twigs: SP>SO>AV>PR>TR.The order of pathogenicity of F.sporotrichioides to the branches of five apple living trees was: Wang Lin > Guoguang > Fuji > Hanfu > wild apple.The order of pathogenicity and aggressive mess of the five Fusarium species to the detached apple leaves was:(1)Wild apple leaves: SP>SO>AV>PR>TR;(2)Fuji leaves: SP>PR>AV>SO>TR;(3)Guoguang leaves: SP>AV>PR>SO>TR;(4)Hanfu leaves: SP>AV>SO>PR>TR;(5)Wang Lin leaves: SP>SO>AV>PR>TR.Under in vitro conditions,there was no difference in the size of the lesions inoculated on the leaves and twigs of different varieties of apples,indicating that the leaves and twigs of Fusarium infecting detached apple materials were not affected by apple tree species.Under field conditions,five species of Fusarium were inoculated by mycelial wound inoculation method,and it was found that F.sporotrichioides can infect the twigs of five apple trees,causing different degrees of xylem browning symptoms.Although the living apple trees did not produce any symptoms after inoculation of five species of Fusarium by the method of spore suspensions,it still indicated that the filed inoculation test was susceptible to varieties of external factors,and it is necessary to explore and repeat the experiment in future.