Identification Of The Pathogen Causing Potato Fusarium Wilt And Verticillium Wilt,and The Study Of Their Co-Infection Process

Potato verticillium wilt caused by V.dahliae and potato fusarium wilt caused by Fusarium spp.are soil borne vascular diseases that affect potato quality and yield,causing serious damage to potato production.In this study,we identified the pathogens of diseased plants of potato were collected from 2018 to 2019 via Koch’s postulate.The agrobacterium tumefaciens mediated transformation(ATMT)system was used to genetically transform both V.dahliae and F.oxysporum strains obtained from a single potato disease sample,and positive transformants of V.dahliae with GFP marker and F.oxysporum with m Cherry marker were obtained.The above positive transformants were used to study the coinfection process of potato.The results obtained are as follows:1.A total of 241 pathogens were isolated from 463 potato diseased samples collected from 42 potato growing areas along the foothills of Yin Mountain from 2018 to 2019.Morphology observations combined with molecular techniques was used for identification,and the results showed that there were 102 V.dahliae strains causing potato verticillium wilt,accounting for 42.3%;139 strains of Fusarium spp.causing potato fusarium wilt,accounting for 57.8%,they are F.acuminatum,F chlamydosporum,F.sambucinum,F.solani,F.proliferum,F.equisteti,F.redolens,F.tricinctum,F.verticilloides and F.oxysporum.There was a total of 26 potato locations where both fusarium strains and verticillium strains were isolated,accounting for 51.2% and 48.8%,respectively.Among the samples collected,10 had both V.dahliae and Fusarium spp.isolated from a single diseased plant and distributed in nine potato fields.2.One representative strain randomly chosen from different species of Fusarium was used to perform biological characteristics study.It is beneficial to the growth of all test strains under the conditions of 25-30 ℃ and 4-11.Different species of Fusarium has significant difference in the growth rate,sporulation,toxicity and pathogenicity.3.10 pairs of Fusarium spp.and V.dahliae isolated and obtained from single potato diseased samples were selected and their co-infection in potatoes was identified via Koch’s postulate.There were some differences between the pathogenicity of the single and coinoculated strains.4.Resistance evaluation of 26 potato varieties against both F.oxysporum and V.dahliae using root-dipping method was carried out.The results showed that 11 potato varieties had different levels of resistance to F.oysporium(LD-X)and V.dahliae(V.d-8),accounting for 42.31%.However,9 potato varieties represented by Zhongshu 19 showed resistance to LD-X and V.d-8,while 6 potato varieties showed susceptibility.5.ATMT was used to genetically transform both V.dahliae and F.oxysporum strains obtained from a single potato diseased sample,and positive transformants of V.dahliae labelled with GFP and F.oxysporium labelled with m Cherry were obtained.Compared transformants with wild type strain for biological characteristics and virulence.The transformants V.d G9-GFP and F.o M4-m Cherry were chosen for subsequent studies.6.Using V.d G9-GFP and F.o M4-m Cherry to observe the co-infection process of potato root,we found that the tip of potato root was the main site of early stage of infection.Conidia could germinate and develop into mycelia expand in the cortical cells and catheter of the root tip.V.d G9-GFP colonized the vascular bundles faster than F.o M4-m Cherry.However,the proliferation rate of F.o M4-m Cherry was significantly faster than that of V.d G9-GFP.This microscopic observation was consistent with the results of q-PCR quantitative assay.7.Using V.d G9-GFP and F.o M4-m Cherry to observe the co-infection process of the above ground tissues of potato,the results showed that the Colonization expansion speed of V.d G9-GFP was faster than F.o M4-m Cherry.The results of this microscopic observation can be reconciled by the results of the q-PCR quantitative assay.8.Co-infection potato using both V.d G9-GFP and F.o M4-m Cherry showed that both V.d G9-GFP and F.o M4-m Cherry were able to colonize the vascular bundles of potato stolons after 35 days of co-inoculation.However,only the green fluorescent signal of V.d G9-GFP or the red fluorescent signal of F.o M4-m Cherry could be observed on tubers harvested from the co-inoculated potato plants.The results from the tuber isolation experiment also confirmed that the two pathogens were not isolated from single potato tuber after co-inoculation.