CN116751803A - Application of VdCreC gene in the inhibition of growth, pathogenicity and carbon metabolism of Verticillium dahliae - Google Patents

Abstract

The invention is applicable to the field of functional gene technology and provides the application of VdCreC gene in the growth of Verticillium dahliae. The application of VdCreC gene in the pathogenicity of Verticillium dahliae is provided. The application of VdCreC gene in inhibiting carbon metabolism of Verticillium dahliae is provided. A medicine for preventing and treating cotton diseases is provided, including a reagent for down-regulating the expression of VdCreC gene or protein. The present invention proves that VdCreC not only plays an important role in inhibiting carbon metabolism, but also affects the growth, development and pathogenicity of Verticillium dahliae. It has important theoretical value for the prevention and control of Verticillium dahliae and the development of new fungicides. It has important practical significance for promoting safe cotton production.

Description

Application of VdCreC gene in growth, pathogenicity and carbon metabolism inhibition of verticillium dahliae

Technical Field

The invention belongs to the technical field of functional genes, and particularly relates to application of a VdCreC gene in verticillium dahliae growth, pathogenicity and carbon metabolism inhibition.

Background

Verticillium dahliae (Verticillium dahliae), belonging to the genus Verticillium fungi, contains many degrading enzymes (Cazymes) that degrade plant cell wall components, including pectinases, cellulases, amylases, etc., and by long-term co-evolution with a host, verticillium dahliae produces different carbohydrate hydrolases according to the polysaccharide component of the host cell wall, whose function confers specific infection characteristics to plants infected by Verticillium dahliae, and some genes encoding carbohydrate hydrolases directly affect the pathogenic ability of Verticillium dahliae to plants. Carbon metabolism inhibition (Carbon Catabolite Repression, CCR) is taken as a global regulatory factor and participates in regulating and controlling the expression of a plurality of carbohydrate hydrolase genes, carbon metabolism inhibition in fungi such as saccharomyces cerevisiae, aspergillus nidulans and the like participates in pathogenicity of pathogenic bacteria, and in addition, the carbon metabolism inhibition genes participate in the cellular processes such as energy metabolism, normal growth and development, carbohydrate metabolism and the like of the fungi, so that the pathogenicity of verticillium dahliae can be directly influenced when the carbohydrate hydrolase related genes are destroyed.

In order to study the function of the carbon metabolism inhibitor genes of Verticillium dahliae, 1 carbon metabolism inhibitor gene was found by searching the Verticillium dahliae genome database under the gene number VDAG-05549, which was designated as VdCrec.

Disclosure of Invention

The embodiment of the invention aims to provide an application of VdCreC gene in the growth of verticillium dahliae, and aims to solve the problems in the background technology.

The embodiment of the invention is realized in such a way that the VdCreC gene is applied to the growth of the Verticillium dahliae.

Preferably, the growth comprises growth rate, propagule yield.

Preferably, the propagules include microsclerotia and conidia.

It is a further object of embodiments of the present invention to provide the use of the VdCreC gene in the pathogenicity of Verticillium dahliae.

Preferably, the pathogenicity is expressed on one or several of the following indicators: index of disease, hyphal penetration ability, and host colonization ability.

It is a further object of embodiments of the present invention to provide the use of the VdCrec gene in the inhibition of carbon metabolism in Verticillium dahliae.

Preferably, the VdCreC gene is involved in glucose-induced inhibition of carbon metabolism by Verticillium dahliae.

It is yet another object of an embodiment of the present invention to provide a drug for controlling cotton diseases, comprising a reagent for down-regulating VdCreC gene or protein expression.

According to the embodiment of the invention, the expression of the VdCrec gene in different time and different tissues is analyzed, and the result shows that the expression quantity of the VdCrec gene in 12d and hypha is highest in the culture of the wild strain V592 in a PDA solid culture medium, a knockout carrier taking the VdCrec as a target gene is constructed according to the homologous recombination principle, the conidium of the cotton verticillium V592 strain is transformed by an agrobacterium-mediated genetic transformation method (ATMT), and 2 VdCrec gene knockout mutants are obtained by screening; meanwhile, an overexpression vector taking the VdCreC gene as a target is constructed, and 2 VdCreC overexpression strain is obtained through screening. Compared with the wild strain V592 and the over-expression strain, the aerial hypha formed by the VdCreC knockout strain is reduced, the growth rate, the spore yield, the spore germination rate and the microscler yield are reduced, and the pathogenicity to cotton is reduced;

the carbon source utilization result shows that VdCrec promotes the utilization of glucose, sucrose, lactose, raffinose and pectin, inhibits the metabolism of xylan, does not influence the utilization of galactose, performs a carbon metabolism inhibition test on VdCrec gene knockout mutant, and after glucose is added into a culture medium taking cellulose and starch as carbon sources, the inhibition rate of VdCrec knockout mutant on the utilization of cellulose and starch is obviously lower than that of wild strain V592, thus showing that VdCrec gene participates in the carbon metabolism inhibition caused by glucose;

in conclusion, the VdCreC gene affects the growth and development and pathogenicity of the verticillium dahliae and plays an important role in inhibiting carbon metabolism.

Drawings

FIG. 1 is a colony morphology of the Verticillium dahliae ΔVdCrec mutant, the complementation strain, and the wild-type strain V592 provided in example 3;

FIG. 2 is a microscopic observation of microsclerotium formation of the Verticillium dahliae ΔVdCrec mutant, the complementation strain and the wild-type strain V592 provided in example 4;

FIG. 3 shows the sporulation measurement results of the Verticillium dahliae DeltaVdCrec mutant, the complementation strain and the wild-type strain V592 provided in example 5;

FIG. 4 is a microscopic observation of the Verticillium dahliae DeltaVdCrec mutant, the complementation strain and the spore-forming stalk of the wild strain V592 provided in example 5;

FIG. 5 shows the pathogenicity determination of the Verticillium dahliae DeltaVdCrec mutant, the complementation strain and the wild strain V592 provided in example 6 on cotton (the upper graph shows the pathogenicity plant morphology; the lower graph shows the disease index result);

FIG. 6 is a graph showing the results of measurement of the penetration ability of the hyphae of Verticillium dahliae ΔVdCrec mutant strain, complementation strain and wild strain V592 provided in example 7 into cotton;

FIG. 7 shows the phenotype of the ΔVdcreC gene mutant provided in example 8, the inhibition ratio of 9d on different carbon source media;

FIG. 8 is an amylase and cellulase activity assay of the ΔVdCrec gene mutant strain provided in example 9.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

In the embodiment of the invention, the nucleotide sequence of the VdCrec gene is shown as SEQ ID NO. 1.

In order to determine whether the VdCrec gene knockout affects the formation of the Verticillium dahliae conidium, the invention constructs a VdCrec gene knockout Verticillium mutant strain delta VdCrec on the basis of a wild type strain V592 and carries out gene overexpression on the basis of the wild type strain V592 to obtain two overexpression strains OE-VdCrec-1, OE-VdCrec-2, and the construction method of the VdCrec gene knockout Verticillium mutant strain delta VdCrec is completed by utilizing the principle of homologous recombination, and can be concretely seen in the prior art to obtain knockout and overexpression mutants (WANG S, XING H, HUA C, GUO H S, ZHANimproved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT) -base gene-disruption method for Verticillium dahliae.Phytopathology,2016,106 (6): 645-652.);

in the embodiment of the invention, delta VdCrec, an over-expression strain OE-VdCrec and a wild strain V592 are respectively taken as experimental objects, and the following indexes are respectively measured: 2 VdCrec knockout mutant strains and 2 overexpression body strains are obtained through agrobacterium-mediated homologous recombination principles, functions of VdCrec genes are explored for the Vdcrec gene mutant strains and the wild strains in biological properties, pathogenicity identification and the like, the VdCrec genes are expressed in verticillium dahliae hyphae, microsclerotium and spores, but the highest expression quantity in microsclerotium indicates that the Vdcrec genes of the verticillium dahliae have tissue expression difference, the time expression difference analysis of the VdCrec genes shows that the Vdcrec genes grow for 16d in the verticillium dahliae PDA culture medium, and the biological property measurement shows that the VdCrec genes influence the colony morphology of the verticillium dahliae and promote the germination and microsclerotium formation of the verticillium dahliae;

the growth conditions of the VdCrec mutant in different carbon sources indirectly reflect the expression conditions of the gene on the corresponding carbon source metabolic enzyme genes, and the inhibition rate of the Vdcrec mutant in a galactose culture medium is found to be no different from that of a wild strain, the inhibition rate of the Vdcrec mutant in glucose, sucrose, lactose, raffinose and pectin culture mediums is obviously higher than that of the wild strain, and the inhibition rate of the Vdcrec mutant in a xylan culture medium is obviously lower than that of the wild strain, so that the Vdcrec gene does not influence the expression of the galactose-related metabolic enzyme genes, the gene expression of the glucose, sucrose, lactose, raffinose and pectin-metabolism-related enzyme genes is positively regulated, and the expression of the xylan-metabolism-enzyme-related genes is negatively regulated, so that different inhibition rates are shown in different carbon source culture mediums;

the pathogenicity identification result shows that the VdCreC gene affects pathogenicity of the verticillium dahliae;

in conclusion, the VdCreC gene affects the mycelium morphology, growth and development and pathogenicity of the verticillium dahliae, and plays an important role in inhibiting carbon metabolism;

according to the embodiment of the invention, the VdCreC gene is knocked out and complementary in function, and the function of the gene in the growth and development of the verticillium dahliae and the carbon metabolism inhibition is researched, so that the VdCreC gene plays an important role in the growth and development of the verticillium dahliae, spore production, pathogenicity and other development aspects, and plays an important role in identifying and regulating and controlling the carbon metabolism inhibition pathway of the verticillium dahliae.

Specific implementations of the invention are described in detail below in connection with specific embodiments.

Example 1, a construction method of a VdCreC gene knockout verticillium dahliae mutant strain Δvcrec:

designing primers according to upstream and downstream homology arms of the VdCreC gene (the nucleotide sequence is shown as SEQ ID NO: 1), constructing a knockout vector, taking a wild strain V592 as an initial strain, constructing a mutant strain, and finally obtaining a Verticillium dahliae mutant strain (delta VdCreC-1, delta VdCreC-2) for knocking out the VdCreC gene;

the specific construction process is as follows:

(1) Amplification of homologous arms of the target genes:

2 pairs of primers are designed by taking the upstream and downstream of VdCrec genes as homologous arms, and the genome DNA of the fallen leaf strain V592 is taken as a template, and I-5 is used ^TM 2 XHigh-Fidelity Master Mix High-fidelity DNA polymerase amplifies the upstream and downstream homology arms of the VdCreC gene, primers VdCreC-s-f and VdCreC-s-r amplify the upstream homology arms of the VdCreC gene, and primers VdCreC-x-f and VdCreC-x-r amplify the downstream homology arms of the VdCreC gene;

PCR reaction system: i-5 ^TM 2 Xhigh-Fidelity Master Mix 25 mu L, vdCreC-s (x) -f/VdCrec-s (x) -r 1 mu L each of DNA and 1 mu L of DNA were added to a 0.2mLPCR tube and ddH was used ₂ O is complemented to 50 mu L system, and the PCR reaction program is as follows: preheating at 98deg.C for 1min, melting at 98deg.C for 15s, annealing at 60deg.C for 15s, extending at 72 deg.C for 15s, maintaining at 72 deg.C for 5min, maintaining at 20 deg.C for 2min to finish reaction, melting to extension stage for 30 cycles, taking3-5. Mu.L of PCR products were detected by gel electrophoresis in a 1% agarose gel electrophoresis with Goldenview added, and observed and photographed under an ultraviolet gel imager. Purifying the target gene fragment of the residual PCR product with correct bands according to the step Gel Extraction Kit of OMEGA biological company product and measuring the concentration;

wherein the upstream and downstream homology arm primers:

VdCreC-s-f

CTTGCTGAGGTCTTAATTAAGGACAGACTCTAGAGGTCAATCC (shown as SEQ ID NO: 2)

VdCreC-s-r

AGTGCTGAGGCATTAATTAACGTCTCGATCATGGGCATGGCTC (shown as SEQ ID NO: 3)

VdCreC-x-f

CCCGCTGAGGACTTAATTAAGTTAAGTTGGGTTCCGACGAG (shown as SEQ ID NO: 4)

VdCreC-x-r

CTCGCTGAGGGTTTAATTAAAGGCCGTCATCAAGGAGTAGAGG (shown as SEQ ID NO: 5)

(2) The knockout vector used in the experiment is pGKO-HPT, and the vector plasmid is subjected to linearization digestion by using PacI, and the system is as follows: mu.L of PacI, 35. Mu.L of vector plasmid, 5. Mu.L of 1 XCutsmart, ddH ₂ O is complemented to 50 mu L, the temperature of a water bath kettle is kept constant, enzyme digestion is carried out for 10-12h, 3-5 mu L of PCR products are taken in the next day, gel electrophoresis detection is carried out in 1% agarose gel electrophoresis added with Goldenview, the detection of enzyme digestion strips is carried out by observing under an ultraviolet gel imager, the target fragments are carefully cut off, the OMEGA biological company product Gel Extraction Kit is used for purifying the linearized carrier fragments, after gel electrophoresis verification again, the purified carrier fragments are split-packed and the concentration is measured, and the purified carrier fragments are stored in a refrigerator at the temperature of minus 20 ℃ for standby, so that repeated freeze thawing is avoided;

(3) In-fusion cloning: connecting an upstream and downstream homology arm of a target gene obtained after amplification and purification and a carrier fragment obtained after linearization and purification by using In-fusion enzyme, wherein the system is as follows: 3 mu L of the homologous arm gene purification products on the upstream and downstream of the target gene, 1 mu L of each of 5 XCE multi s Buffer and Exnase multi s and 2 mu L of linearization carrier are placed in a 37 ℃ water bath kettle for connection for 30min, placed on ice for subsequent experiments or temporarily stored in a-20 ℃ refrigerator, and subjected to heat shock to transform escherichia coli and extract recombinant plasmids;

(4) Agrobacterium-mediated genetic transformation: electric shock transformation of agrobacterium: taking out recombinant plasmid and agrobacterium competent cells from-20deg.C refrigerator and-80deg.C refrigerator respectively, and rapidly thawing on ice; taking out 1 mu L of recombinant plasmid, adding the recombinant plasmid into the agrobacterium competent cells, and standing on ice for 10min; wiping off the water on the wall of the sterilized and precooled electric shock cup, adding the mixed solution, and then placing the electric shock into an electric shock instrument for instantaneous electric shock; adding 500 mu L of LB liquid medium without any antibiotics, blowing and mixing by a pipettor, sucking the mixed liquid in a electric shock cup, placing in a 1.5mL centrifuge tube, and shaking and culturing at 200rpm in a shaking table at 28 ℃ for 45min; instantaneously centrifuging, discarding 500 mu L of supernatant, fully mixing the rest bacterial liquid by a pipettor, uniformly coating the bacterial liquid on an LB solid culture medium containing Kan and Rif antibiotics in an ultra-clean bench, placing the LB solid culture medium in a constant temperature incubator at 28 ℃ for dark culture for 2-3 d, screening positive transformants by PCR, shaking a colony with correct PCR to obtain the agrobacterium bacterial liquid, taking 100 mu L of the agrobacterium bacterial liquid into 10mL of IMAS (Kan-containing) liquid culture medium, shaking and culturing at 200rpm in a shaking table at 28 ℃ until the OD600 value is about 0.5, thawing the collected verticillium dahliae conidium on ice in advance, and uniformly mixing the conidium of verticillium dahliae and agrobacterium according to the proportion of 1:1; uniformly coating 200 mu L of the mixed solution on an IMAS solid culture medium paved with sterilized bacteria, repeating for 3 times, wherein only a plate coated with the conidium of Verticillium dahliae is used as a positive control, and only a plate coated with the bacterial solution of the recombinant plasmid of the agrobacterium is used as a negative control; after 48h, the filter paper is removed from the IMAS solid medium and placed on a PDA (containing HygB+Cef+Car+F2dU) plate, the transformants are grown in dark at 26 ℃ for 5-7 d, single fungus colonies are picked on an ultra-clean workbench, streaked and inoculated on the PDA (containing Cef+HygB) medium, the fungi are grown in dark at 26 ℃ for about 10d, the growth condition of the fungi in the plate is always observed, only homologous recombination knocked-out transformants grow on the resistant plate, false positive transformants do not grow, and after the positive transformants grow out, single spore separation and subsequent test are carried out on the positive transformants, and finally two knocked-out transformants are obtained.

Example 2, a method for constructing a Verticillium dahliae VdCrec gene overexpression OE-VdCrec:

the method comprises the steps of taking a verticillium dahliae wild type V592 as an initial strain, obtaining a verticillium dahliae VdCrec gene overexpression body (OE-VdCrec), using a vector p1300-Neo-oLiC-Cas9-TtrPC, and carrying out tangential arrangement on 50 mu L of an XbaI and BamHI double enzyme digestion system by using XbaI and BamHI double enzymes: p1300-Neo-oLiC-Cas9-TtrPC 20. Mu.L, xbaI 1. Mu.L, bamHI 1. Mu.L, 1 XMbus buffer 2.5. Mu.L, ddH ₂ O ₂ 5.5 mu L, and carrying out enzyme digestion at 37 ℃ overnight, and then cutting the gel on the next day to recover the target strip. UsingII One Step Cloning Kit construction of an overexpressed recombinant plasmid, wherein p1300-Neo-oLiC-Cas9-TtrPC and VdCrec genes are connected by ExnaseII enzyme, and a single-segment connection system of p1300-NeO-LiC-Cas9-TtrPC is as follows: 5 XCEIIbuffer 2. Mu.L, p1300-Neo-oLiC-Cas9-TtrPC 200ng, 80ng of the gene fragment of interest, exnaseII 1. Mu.L, ddH ₂ O is added to 10 mu L, and the mixture is connected for 30min at 37 ℃;

the remaining methods and the obtaining of the knockout mutant were identical, but differ from the obtaining of the knockout positive transformant in that the knockout mutant was obtained by 1.0X10 of conidia of the wild-type V592 of Verticillium dahliae ⁶ Conidia/mL and Agrobacterium containing knockout vector were mixed in equal proportions, and overexpressing mutant positive transformants were obtained by 1.0X10% V592 conidia ⁶ Mixing the agrobacterium containing the over-expression vector in equal proportion in the concentration of concdia/mL; secondly, the first screening culture medium of the positive transformants of different complementary mutants of the resistance screening culture medium is PDA+HygB+Cef+Tim+G418, and the second screening culture medium is PDA+Cef+G418.

Example 3 application of VdCreC Gene in Verticillium dahliae growth:

(1) Determination of colony growth Rate

Culturing with the VdCreC gene knocked-out verticillium mutant strain prepared in example 1, the VdCreC gene overexpression mutant prepared in example 2 and the wild strain V592 as experimental objects, inoculating the selected mycelium in the center of a PDA culture medium, culturing in darkness at 22 ℃, measuring the colony diameters of all strains on the 5 th day and the 9 th day after inoculation, and calculating the average growth rate of the colonies according to the following formula:

average growth rate of colony = colony growth rate = (9 th d average colony growth diameter-5 th d average colony growth diameter)/4;

3 replicates were set for each strain and the colony morphology was recorded by photographing on day 15;

(2) Morphological observation of hyphae

Culturing different strains of Verticillium dahliae on PDA plate by streaking, obliquely inserting sterilized cover glass into streaking position, culturing at 22deg.C for 3d, taking out cover glass, and observing hypha growth condition under microscope;

as a result, as shown in FIG. 1, it can be seen from FIG. 1 that the wild-type strain V592 formed a large amount of black microsclerotia on the PDA plate with white aerial hyphae, but that 2 VdCreC gene knockout strains produced less white aerial hyphae than the colony edge of the wild-type strain V592, indicating that VdCreC affected the growth phenotype of Verticillium dahliae.

Example 4 determination of microsclerotia:

the VdCreC gene-knocked-out verticillium dahliae mutant strain prepared in example 1, the VdCreC gene-overexpressed mutant prepared in example 2 and the wild strain V592 were used as subjects for cultivation to obtain a bacterial cake. Respectively taking about 10 bacterial cakes from each bacterial strain by using a puncher, inoculating the bacterial cakes into a Charles (containing kan) liquid culture medium, and shaking and culturing for 3-5 d in a shaking table at 26 ℃ at 200 r/min; collecting conidium by filtration; regulating spore concentration to 1.0X10% conidium concentration ⁶ CFU/mL, 100. Mu.L of MM plate (NaNO) uniformly coated on flat glass paper was pipetted ₃ 2g、KH ₂ PO ₄ 1g、MgSO ₄ ·7H ₂ O0.5 g, KCl 0.5g, citric acid 10mg, znSO ₄ ·7H ₂ O 10mg、FeSO ₄ ·7H ₂ O 10mg，NH ₄ Fe(SO ₄ ) ₂ )·12H ₂ O 2.6mg，CuSO ₄ ·7H ₂ O 0.5mg，NnSO ₄ ·H ₂ O 0.1mg，H ₃ BO ₃ 0.1mg，Na ₂ MoO ₄ ·2H ₂ O0.1 mg, glucose 2g,1.5 agar, add distilled water to 1L. Sterilizing at 120deg.C under high pressure for 20 min), culturing at 22deg.C in dark for 15d, photographing, observing, and scraping glass paperWeighing and measuring the culture, recording the wet weight of the culture, standing the microsclerotium at room temperature for 48 hours, airing the microsclerotium, weighing the microsclerotium on a balance, and recording the dry weight data of the microsclerotium;

as a result, as shown in FIG. 2, it was found from FIG. 2 that 2 VdCrec gene knockouts were not significantly changed from the wild-type strain V592, all strains produced black microsclerotia, the microsclerotia yield of 2 VdCrec gene knockouts was significantly lower than that of the wild-type strain V592, and the microsclerotia yield of 2 Vdcrec gene overexpression strains were not significantly different from that of the wild-type strain V592.

Example 5 to determine whether a VdCreC gene knockout affects the formation of conidia of verticillium dahliae, a specific assay was as follows:

the concentrations were 1.0X10 each ⁶ CFU/mL knockout mutant (strain obtained by construction in example 1), overexpressing strain (strain obtained by construction in example 2) and V592 were inoculated into Czapek-Dox liquid medium, shake-cultured at 26℃under 200r/min for 5d, isolate spores, and aspirate 1.0X10 s ⁶ 100 mu L of CFU/mL spore suspension is inoculated in Charles (containing kan) culture medium, 3 biological repeated experiments are set for each strain, 1mL of bacterial liquid is sucked every 24 hours in the process of shaking culture at the speed of 200r/min in a shaking table at the temperature of 26 ℃, and the spore concentration is measured by a blood cell counting plate and recorded for 7 days;

as a result, as shown in FIGS. 3 and 4, it can be seen from FIG. 3 that the spore yield of 2 VdCrec gene knockouts was significantly lower than that of the wild-type strain V592, and that of 2 VdCrec over-expressed strain was significantly higher than that of the wild-type strain V592, starting from FIG. 5 d. The VdCreC gene is explained to promote the spore production of verticillium dahliae; as can be seen from FIG. 4, when the mutant was observed under a microscope, the DeltaVdCrec mutant produced little wheel-branched pedicel compared to the V592 strain.

Example 6 in order to clarify the effect of VdCreC gene knockout on verticillium dahliae pathogenicity, the pathogenicity of VdCreC gene knockout mutant on cotton was determined with wild-type strain V592 and an over-expressed strain as controls, and the pathogenicity was determined as follows:

the knock-out mutant, the over-expression strain and V592 constructed as aboveInoculating to Czapek-Dox liquid culture medium at 26deg.C under shaking culture at 200r/min for 5d, and inoculating 200mL of cotton with concentration of 1.0X10 by root soaking inoculation method ⁷ The CFU/mL bacterial liquid is repeated for 3 water planting boxes (36 cotton seedlings) of each strain, the bacterial liquid is observed every day after inoculation, the disease is counted from the beginning of the disease, the disease is generally recorded to be one month after the disease every 3d, and the disease grading standard is as follows: level 0: does not cause disease; stage 1: 1-2 pieces She Fabing; 2 stages: 1 leaf of true leaf; 3 stages: 2 pieces of true leaves are ill; 4 stages: 3 or more than 3 true leaves are diseased, and the disease index is calculated according to the following formula:

disease index= [ Σ number of plants at each stage×number of stages/(total number of plants×highest disease stage) ]×100;

the disease index is the repeated average value of 3 biological experiments;

as a result, as shown in FIG. 5, it can be seen from FIG. 5 that compared with the wild-type strain V592, the cotton inoculated with the VdCreC knockout strain exhibited symptoms of leaf yellowing, wilting and scorch after 3 days, and the onset symptoms at 25d were significantly lower than those of the wild-type strain, and the disease index of the VdCreC knockout strain was also significantly lower than those of the wild-type and overexpresser strains.

Example 7 to analyze whether VdCreC gene knockdown resulted in a decrease in verticillium dahliae virulence associated with its ability to penetrate the host, cellophane penetration experiments were performed on each of the knockdown mutant strains with V592 as a control, as follows:

spreading glass paper with the size basically consistent with that of the culture dish after high-temperature sterilization treatment on the poured MM basic culture medium, picking hypha by using a toothpick, inoculating the hypha to the center of the glass paper, respectively culturing for 3d, removing the glass paper, allowing strains to grow for 7d again, observing whether colonies can grow on the culture dish, and setting 3 repetitions for each strain;

the results are shown in FIG. 6, and according to FIG. 6, it can be seen that the VdCreC gene knockout mutant can penetrate the cellophane when inoculating 3d, and the results show that VdCreC gene knockout does not cause the loss of the penetrability of the Verticillium dahliae on the cellophane.

Example 8, verticillium dahliae produces 514 carbohydrate hydrolases, of which 152 are related to pectin, 92 are related to cellulose, 69 are related to xylan and 61 are related to starch, and the regulation and control effects of VdCrec genes on different carbon source hydrolases can be indirectly reflected by researching the utilization condition of VdCrec gene knockout mutants of verticillium dahliae, so that each VdCrec mutant strain and wild type strain V592 are inoculated on different carbon source culture mediums to observe the growth condition of the strain;

as shown in fig. 7, it can be seen from fig. 7 that 2 VdCreC gene knockout strains produced less black microsclerotia in glucose, xylan, pectin medium than the wild-type strain V592, and that the phenotype on the medium without additional carbon source, galactose, lactose, raffinose, and xylan carbon source was not significantly different from the wild-type strain V592, and the measurement of colony diameter showed that the inhibition ratio analysis: compared with the wild type strain V592, the inhibition rate of 2 VdCreC gene knockout body strains in a culture medium taking glucose, sucrose, lactose, raffinose and pectin as carbon sources is obviously increased, the inhibition rate of 2 VdCreC gene knockout body strains in a culture medium taking xylan as a carbon source is obviously reduced, and the inhibition rate in a culture medium taking galactose as a carbon source is not obviously different from that of the wild type strain V592; compared with the wild type strain V592, under the condition of taking galactose as a carbon source respectively, the inhibition rate of the 2 VdCrec gene overexpression body strains has no obvious difference with the wild type strain; under the condition that xylan is taken as a carbon source, the inhibition rate of 2 VdCrec gene overexpression body strains is obviously reduced, and under the condition that glucose, sucrose, lactose, raffinose and pectin are taken as carbon sources, the inhibition rate of 2 VdCrec gene overexpression body strains is obviously increased, so that in conclusion, vdCrec genes do not influence the expression of galactose-related enzyme genes, the gene expression of glucose, sucrose, lactose, raffinose and pectin metabolism-related enzymes is positively regulated, and the expression of xylan metabolism-related enzymes is negatively regulated, so that different inhibition rates are shown in different carbon source culture mediums.

Example 9, primary task of carbon metabolism inhibition was to sensitively identify a preferred carbon source, thereby regulating other secondary carbon sources, inoculating each mutant strain and wild strain of VdCreC to a medium containing starch, starch+glucose, cellulose, cellulose+glucose as carbon sources, respectively staining with iodine solution and congo red, measuring the size of the transparent circle, and calculating the inhibition rate in the presence of glucose;

the results are shown in FIG. 8, and according to FIG. 8, the phenotype of each strain of VdCrec gene on each carbon source culture medium is not obviously different from that of the wild strain V592, under the condition of glucose, 2 VdCrec gene over-expression strains and the wild strain V592 lose the utilization of starch and cellulose, no transparent circle is generated, namely, starch and cellulose are not decomposed, so that the glucose has an inhibition effect on the utilization of starch and cellulose, and the inhibition rate is 100%; in the presence of glucose, the 2 VdCrec gene knockout body strains can still observe obvious transparent rings, which shows that VdCrec gene knockout releases the inhibition effect of glucose on starch and cellulose metabolism, but promotes the utilization of starch and cellulose, the inhibition rate of starch utilization reaches 6.37% and 8.77%, and the inhibition rate of cellulose utilization reaches 19.15% and 22.80%, respectively, and in conclusion, vdCrec gene participates in carbon metabolism inhibition response caused by glucose in Verticillium dahliae.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (8)

1. Application of VdCreC gene in the growth of verticillium dahliae.
2. 2. The use according to claim 1, wherein the growth comprises growth rate, propagule yield.
3. 3. The use according to claim 2, wherein the propagules include microsclerotia and conidia.
4. Application of VdCreC gene in verticillium dahliae pathogenicity.
5. 5. The use according to claim 4, wherein the pathogenicity is expressed on one or several of the following indicators: index of disease, hyphal penetration ability, and host colonization ability.
6. Application of VdCreC gene in carbon metabolism inhibition of Verticillium dahliae.
7. 7. The use according to claim 6, wherein the VdCreC gene is involved in glucose-induced carbon metabolism inhibition of verticillium dahliae.
8. 8. A cotton disease control drug, comprising an agent that down regulates VdCreC gene or protein expression.