| Fusarium oxysporum f.sp.cucumerinum(Foc)is a fungal pathogen that causes cucumber Fusarium wilt disease,one of the most important diseases to reduce yield and quality of cucumber.Continuous cropping(monoculture)in greenhouse cucumbers by resistant cultivars is not sustainable in long-term basis due to yield reduction and encourages virulence of Fusarium oxysporum.Virulence variation of Fusarium oxysporum is usually associated with the differentially expressed virulence related genes that are located on accessory or lineage-specific(LS)chromosomes.Recent research results showed that virulence of a weakly virulent strain of Foc,Foc-3b(wild type,WT)was significantly increased after serial passage on moderately resistant cucumber cultivar(ZN106)upto fourth generations,but decreased on susceptible cucumber cultivar(ZN6).Based on findings,a weakly virulent(WT～Foc-3b)and its induced virulence variation(InVir～Foc-Ra-4)strain obtained from fourth generations on moderately resistant cultivar termed as highly virulent(Foc-Ra-4)strain of Foc were selected in this research work.For understanding the virulence differentiation of Foc in between strains(WT and InVir),experiments were performed in laboratory as well as greenhouse conditions.Firstly,investigate the growth rates,virulence and stress responses between strains.Secondly,transfer the green fluorescent protein(GFP)reporter gene in both strains and observe the infection processes and colonization patterns in susceptible(ZN6)and moderately resistant(ZN106)cucumber cultivars,and also analyze the expression of virulence related genes during in vitro and in planta colonization.Thirdly,focus on the functions of potential virulence genes namely FocSIX11(an effector/SIX11 protein)and FocEl(a hypothetical protein likely effector)in WT strain of Foc through gene deletion and complementation approaches.The main research results were as follows:1.There was a significant variation between strains in terms of growth and conidiation rates,conidial germination,virulence and stress responses.Due to successive inoculation on resistant cultivar(ZN106)by WT strain,the differences were significant in each parameter in between strains.Highly virulent strain exhibited the severe infection on cucumbers than weakly virulent strain.2.Green fluorescent protein(GFP)reporter gene was successfully tagged in both strains of Foc.Highly virulent strain colonized the seedling roots as well as stem bases and stems of both cultivars as much faster than weakly virulent,and blocked/plugged xylem vessels by multiply profusely the fungal structures and therefore,no movement of waters and mineral nutrients from roots to upwards through xylems resulting severe wilt symptoms and finally death of plants.Moreover,the expression levels of six genes namely Unigene6293,Unigene11126(FocSIX11),Unigene7476,Unigene2075,Unigene2791(FocE1)and Unigene16837 were quantified through qRT-PCR analysis from in vitro and in planta samples by the Foc strains.Among them,expression of FocSIX11 in InVir was higher at initial stage of root infection than WT strain.The InVir strain faster secreted in vascular systems(xylem vessels)of cucumber roots at initial stage indicating an increased virulence.Thus,SIX genes or effectors expression at initial stage and primodial ring in the xylem vessels of crown region(stem forming area)are major blocking areas,and shown drastic effects by the vascular/Fusarium wilt causing fungi.Based on qRT-PCR results adjusted with the validation of transcriptome data,FocSIX11 and FocE1 were selected for functional verifications from draft genome sequence of Foc.3.FocSIX11 protein was single copy in the genome with cds 336 bp in length and encodes in 111 amino acids(aa),and homologs of other SIX11 genes of Fusarium oxysporum formae speciales.FocSIX11 protein contains a signal peptide(19 aa),a SCOP domain(63 aa),and a catalytic domain(21 aa)of calcium binding epidermal growth factor(Ca-EGF)demonstrating virulence characters.FocSIX11 expression was up-regulated at 24 and 120 hours after root inoculation in contrast of control(in vitro,0 hour).Gene deletion mutant(FocΔSIX11)showed significantly reduction in conidiation,conidial germination,and increased sensitivity to oxidative stress(hydrogen peroxide,H2O2),cell membrane(sodium dodecylsulphate,SDS)and cell wall(congo red,CR;calcoflour white,CFW)damaging agents,whereas decreased to osmotic stress agent(NaCl).Virulence of deletion mutant significantly reduced on susceptible cucumber cultivar compared with wild type(WT)or complementation mutant(CM)strains.Therefore,FocSIX11 gene involved in Foc virulence during cucumber infection.4.FocEl(a hypothetical protein like effector behaviour),cds 723 bp in length and encodes 240 amino acids with no conservative domain,and homologs of hypothetical or uncharacterized proteins of Fusarium oxysporum formae speciales.FocE1 expression was up-regulated at 24 and 120 hours after root inoculation in compare with control(in vitro,0 hour).The gene deletion mutant(ΔFocE1)displayed significantly reduction in conidiation,conidial germination,and decreased sensitive to osmotic stress agents(NaCl,KCl,glycerol)whereas increased to oxidative stress(H2O2),cell membrane(SDS)and cell wall(CR,CFW)damaging agents,respectively.Virulence of deletion mutant significantly reduced on susceptible cucumber cultivar compared with WT or CM strains.Thus,FocE1 plays a critical role in Foc virulence during infection on cucumber.Cell wall integrity,osmotic and oxidative stresses are involved in Foc virulence and its differentiation.Further investigations will be worthy and interesting to know which molecular mechanisms involved in FocSIX11 and FocE1 genes for apparently decreased in virulence during cucumber infection.To sum up,through functional verification of virulence related genes especially effectors from this research provides an idea about virulence differentiation of Foc during cucumber infection and insights underlying mechanisms of virulence,and also helps to develop innovative ways for the management of cucumber Fusarium wilt. |
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