| Lycium barbarum root rot is a serious disease,which is difficult to control and has complex causes.Pathogenic fungi are considered to be the main factor.L.barbarum has a long history of cultivation in Ningxia.In recent years,cultivars are constantly updated,and different varieties have different susceptibility to root rot Therefore,it is necessary to carry out systematic research on pathogens of different varieties.In this study,the rotting root samples of three cultivars,Ningqi-1,Ningqi-5 and Ningqi-7,were collected from Zhongning,Ningxia.The rot root fungi were obtained and identified based on tissue separation method,and then the pathogens were determined through pot experiments according to Koch’s law.Further research was conducted on the dynamic changes of the dominant pathogen Fusarium solani in the rhizosphere soil of L.barbarum using quantitative real time PCR technology,and the cytological characteristics of its invasion into L.barbarum roots were observed using electron microscopy technology.The main research findings are as follows:1.A total of 136 strains of fungi were isolated from all root rot samples.According to morphological characteristics and molecular identification using conservative sequences,the 35 fungi isolated from Ningqi-1 root rot samples include 19 strains of F.solani,6 strains of Earliella scabrosa,2 strains of F.incarnatum,Mortierella alpina and Alternaria alternata,1 strain of Microdochium bolleyi,Clonostachys rosea,A.tenuissima,and A.brassicae.The 65 fungi isolated from Ningqi-5 root rot samples,include 33 strains of F.solani,18 strains of F.oxysporum,9 strains of E.scabrosa,2 strains of Penicillium pimiteouiense,1 strain of Rhizotonia solani,Acremonium persicinum,C.rosea and F.andiyazi.The 36 fungi isolated from Ningqi-7 root rot samples include 15 strains of F.solani,10 strains of F.oxysporum,4 strains of E.scarbrosa,3 strains of F.redolens,2 strains of C.rosea,1 strain of Penicillium soli and P.raperi.Among them,the isolation frequencies of F.solani in the rotten root of Ningqi-1,Ningqi-5,and Ningqi-7 were the highest,reaching 54.29%,50.77%,and 41.67%,respectively.2.According to Koch’s law,24 representative strains of root rot fungi from different species and genera were selected for pathogenicity identification.It was observed that 8 strains of fungi made the plants show symptoms of root rot.In the symptomatic plants,the leaves of the plants turned yellow,gradually wilt and scorched until they fell off.The roots turned black and rotted,and the cortex fell off and was easy to peel off.The symptoms are consistent with the phenomenon of disease in the field.16 fungi did not cause disease symptoms of root rot in L.barbarum.Isolation,purification,and identification of the fungi from the rotten roots of the diseased plants caused by the 8 strains were carried out again,and the pathogens were identified based on the Koch’s law.F.solani and F.incarnatum were identified as the pathogens of the root rot disease of Ningqi-1 L.barbarum.F.oxysporum,R solani and F.solani were identified as the pathogens of the root rot disease of Ningqi-5 L.barbarum.F.oxysporum,F.solani and F.redolens were identified as the pathogens of the root rot disease of Ningqi-7 L.barbarum.This is the first report of F.redolens causing root rot of Chinese wolfberry in China.3.The Lys2 gene was screened and identified as a specific gene of the dominant pathogen F.solani based on real time quantitative PCR amplification.With the genomic DNA of rhizosphere soil used as templates and Fs-F/Fs-R used as specific primers,the copy number of Lys2 gene were determined to characterize the pathogen F.solani.The results showed that the copy number of Lys2 gene gradually increased with inoculation time,and the growth was slow within 1 to 7 days,with rapid growth within 14 to 21 days.After inoculation with F.solani,scanning electron microscopy and transmission electron microscopy were used to observe the microscopic changes in the root morphology of L.barbarum.On the first day,the pathogen hyphae adhered to the root surface.On the 7th day,a large number of hyphae invaded the periderm,while a small amount of hyphae invaded the phloem.On the 14th and 21st days,the phloem parenchyma cells were filled with a large number of hyphae,causing the cell wall being loose and broken,the cells disintegrated,and the root surface being seriously concave,broken and hollow.On the 28th day,the hyphae infected the xylem.The transmission electron microscope observed that the pathogen accumulated in the cell wall,cell membrane and intracellular,and the infected cell wall turned thickened.The severe cracking of starch grains ultimately leaded to fragmentation and disintegration,which was the main site for F.solani to absorb nutrients and colonize.In conclusion,this study isolated and identified the fungi of rotten root from three main cultivars Ningqi-1,Ningqi-5 and Ningqi-7 in Ningxia,and identified five pathogens of root rot of L.barbarum in Ningxia,which were F.oxysporum,R solani,F.solani,F.incarnatum and F.redolens.Among them,due to the widespread pathogenicity and highest isolation frequency,F.solani was identified as the dominant pathogen of Ningxia L.barbarum root rot disease.It stably proliferates in the rhizosphere soil,quickly identifies and adheres to the root surface,passes through the periderm,absorbs nutrients in the phloem,and rapidly propagates,thereby expands to form a certain infected area.This study provides a theoretical reference for the pathogenesis and prevention and control of L.barbarum root rot disease. |
