Abstract: Agrobacterium tumefaciens Mediated Transformation (ATMT) was used to establish an insertional mutant library. Firstly, we have developed and optimized ATMT system using defoliation strain of V. dahlia, which is the causal agents of wilt on cotton. Furthermore, a library consisted of 4890 T-DNA insertion mutants were generated. Compared with wild-type strain, 34.7% of mutants significantly reduced their colony growth rates among the 196 mutants that were randomly selected, while 34.8% of mutants increased their colony growth rate, 16.8% of mutants lost their ability to produce microsclerotia, 30.0% of mutants decreased their sporulation ability and 27.1% of mutants reduced their pathogenicity. Especially, mutant VdAG30, VdAG153, VdAG151 and VdAG95 showed significant reduce in pathogenicity. In this study, the constructed ATMT mutant library was found to be effective and suitable for defoliating V. dahliae strain and could lay a foundation for cloning candidate pathogenicity-related genes and development-related genes in V. dahliae in the future.
INTRODUCTION
Cotton (Gossypium sp.) is an important economic crop and it is grown in more than 80 countries, especially upland cotton (G. hirsutum L.), which produces more than 95% of the annual cotton crop world wide (Chen et al., 2007) and provides the natural fiber for textile industry (Thibodeaux and Evans, 1986; Wakelyn et al., 2010). China is the world largest cotton producer and it is estimated that 32.0 million bales of cotton would be produced in 2013/14 (Johnson et al., 2014), however, more than 2 million ha of cotton crop was under the attack of Verticillium wilt that caused about 10-15% yield loss due to such a disease (Johnson et al., 2014). Cotton Verticillium wilt is one of the most important diseases and difficult to control (Fradin and Thomma, 2006). Schnathorst and Mathre (1966) first showed that different levels of pathogenicity of V. dahliae isolates from cotton existed in California and classified them as defoliating and non-defoliating pathotypes according to their ability to completely defoliate cotton leaves or to cause only wilt symptoms and no defoliation on cotton cultivars Tanguis (G. barbadense), Acala 4-42 and Delta Pine 15 (DPL) (G. hirsutum), respectively. Since then, the defoliating strains of V. dahliae of cotton were reported in Spain (Perez-Artes et al., 2000; Mercado-Blanco et al., 2003), Turkey (Dervis et al., 2010), Italy (Colella et al., 2008), Tunisia (Bellahcene et al., 2005) and Egypt (El Said et al., 2012). In China, defoliating type of V. dahliae from cotton was first reported in 1983 in Jiangsu Province (Lu et al., 1983) and it had increased up to 83.8% by 2008 (Lin et al., 2012). A similar situation was reported in Xinjiang Autonomous Region, the largest cotton growing region in China, where the defoliating type proportion in Xinjiang was reported to be 39.4 and 33.3% in 2011 and 2012, respectively (Han et al., 2011, 2012).
Therefore, understanding the molecular mechanism between the defoliating type of V. dahliae and cotton interaction has the fundamental importance to design novel control strategies for Verticillium wilt. An effective way to identify pathogenicity or virulence genes is adopted by random mutagenesis, following to screen virulence-reduced mutants on a particular host plant (Jeon et al., 2007). Transferring DNA (T-DNA)-based random insertional mutagenesis provide a unique opportunity to generate genetic mutations in a manner that facilitates subsequent isolation of gene identification (Michielse et al., 2005; Jeon et al., 2007). Using genome the sequences of V. dahliae will be useful for easy identification of T-DNA insertion loci based on detailed genetic blueprints of these pathogens and the potential pathogenicity factors.
Agrobacterium tumefaciens Mediated Transformation (ATMT) has been confirmed to be a useful method to obtain insertional mutant libraries and to identify pathogenicity-related genes. Several publications (Fradin and Thomma, 2006; Gao et al., 2010; Klosterman et al., 2011; Maruthachalam et al., 2011; Tzima et al., 2011, 2012) had identified some V. dahliae pathogenicity and virulence genes. A mitogen-activated protein kinase gene, VMK1, was approved to have a role in pathogenicity (Rauyaree et al., 2005). One glutamic acid-rich protein 1 (VdGARP1), a hydrophilic protein, was achieved through T-DNA insertional mutagenesis library of V. dahliae strain V592 and identified to play a role in pathogenicity (Gao et al., 2010). Maruthachalam et al. (2011) identified several genes involved in pathogenicity on V. dahliae strain VdLs17 from lettuce through ATMT insertional mutagenesis: VdEg-1, VdHMGS, VdMFS1 and VdGPIM3. Tzima et al. (2012) presented that VGB disruption mutant of V. dahliae race 1 of tomato severely impaired virulence on tomato and eggplant. Alternative approaches are needed to address the genetic complexity of this fungus in order to identify the genes that cause the pathogenicity.
In this study, we aim to (1) Optimize conditions of ATMT for a large number of transformants carrying a single copy of T-DNA, (2) Establish a mutant library of a defoliating V. dahliae strain XJ2008 from cotton, (3) Evaluate the library and (4) Get pathogenicity related mutants.
MATERIALS AND METHODS
Fungal and bacterial strains: A defoliating strain XJ2008 of V. dahlia was isolated from a Verticillium wilt of cotton plant sampled in 2008 in Xinjiang Autonomous Region, China. Potato Dextrose Agar (PDA) medium was used to grow XJ2008 and its transformants. Agrobacterium tumefaciens strains AGL-1 and EHA105 were used in this study. The pCT-Hyg vector carrying hygromycin B phosphotransferase gene (Hyg) under control of Aspergillus nidulans trpC promoter was used throughout this study (Liu et al., 2013).
Sensitivity of V. dahliae and A. tumefaciens to hygromycin B: The wild type strain XJ2008 of V. dahliae was inoculated onto PDA plates containing hygromycin B 25, 50, 75, 100 and 150 g mL-1 and without containing hygromycin B as the control and then plates were cultivated at 25°C for 7 days. The sensitivity of V. dahliae to hygromycin B was evaluated by mycelium growth. A. tumefaciens strain AGL-1 and EHA105 were grown on YEP (Yeast Extract and Peptone) plates with different concentrations of cefotaxime sodium as 100, 200 and 300 g mL-1. The experiment was repeated three times and each replicate had three plates.
Agrobacterium tumefaciens mediated transformation: The ATMT referred to the method described by Maruthachalam et al. (2008) was followed with some modifications. Two different types of A. tumefaciens strains i.e., AGL-1 and EHA105 harboring the pCT-Hyg were streaked onto YEP medium plate (10 g L-1 bacto peptone, 10 g L-1 yeast extract, 0.5 g L-1 NaCl and 1.5 g L-1 agar pH 7.0) containing 50 g mL-1 kanamycin (Kan) and 50 g mL-1 rifampicin (Wang et al., 2013) and cultivated at 28°C for 2 days. The single colony was transferred into a 10 mL test tube with 5 mL of Minimal Media (MM) (Adachi et al., 2009) amended with 5 g mL-1 thiamine and 5 g mL-1 of kan and cultivated at 28°C with 220 rpm for 48 h in an orbital shaker. The culture was collected through centrifugation at 12000 rpm for 1 min, then the supernatant was discarded and the pellet was re-suspended in 5 mL IM medium (Bundock et al., 1995) and it re-suspended in fresh IM medium containing acetosyringone (AS) (200 mol L-1) at OD600 = 0.15, after cultivated for 6-14 h at 28°C with 220 rpm to an OD600 of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2. The conidial suspension of V. dahliae contained 1×106 conidia per milliliter and cells of A. tumefaciens were mixed (1:1, v/v) in a 1.5 mL eppendorf tube and kept at room temperature for 5 min. Then 200 μL of this mixture was spread on the co-cultivation plates and cultured following five different co-cultivation times as 12, 24, 36, 48 and 60 h on a cellophane in three different temperature treatments as 23, 25°C and 28°C. The cellophane was transferred onto a new PDA plates augmented with 200 g mL-1 of cefotaxime and 50 g mL-1 of hygromycin B using sterile forceps and cultivated for 5-7 days. Typically, the colonies that could grow on the new PDA plates containing 50 g mL-1 hygromycin B were assumed as putative mutants. And the positive transformants were then stored in 25% (v/v) sterilized glycerol at -80°C until use.
Optimization of ATMT conditions for V. dahliae: We optimized the conditions of ATMT using two different types of A. tumefaciens strains AGL-1 and EHA105, the association parameters were systematically investigated and optimized, including the concentration of Agrobacterium and AS, co-cultivation duration and co-cultivation temperature.
DNA manipulation: Genomic DNA from powdered mycelium of individual transformants and wild-type strain XJ2008 were extracted based on a procedure established by Moller et al. (1992). For Southern blot analysis, genomic DNA was digested with EcoRI, which cuts once in the T-DNA. Electrophoresis and blotting followed standard protocols (Sambrook and Russell, 2001). PCR was conducted with specific primer pair Hyg-F (5’-CCT GAA CTC ACC GCG ACG TC-3’) and Hyg-R (5’-CTA TCC TTT GCC CTC GGA CGA GTG-3’) using a volume of 25 L. The PCR program was as follows: 5 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C and 10 min at 72°C for the final extension. The PCR production was separated by electrophoresis on a 1% agarose gel, stained with Ethidium Bromide (EtBr) and photographed.
Mitotic stability of transformants: To evaluate the stability of inserted T-DNA in transformants, hygromycin B resistant colonies on selection plates were randomly selected and cultured on PDA without hygromycin B at 25°C for 7 days. New mycelia from the colony edge were punched and inoculated onto a fresh PDA plate without hygromycin B following five successive generations. Subsequently, the mycelium from each transformant was inoculated for growth onto the fresh PDA amended with 50 g mL-1 hygromycin B to determine its mitotic stability (Dos Reis et al., 2004; Michielse et al., 2005; Maruthachalam et al., 2011).
Assays for biological characteristics of transformants: The wild-type strain XJ2008 and transformants were inoculated onto PDA plates and cultivated in dark at 25°C for 7 days. The growth rates of transformants were calculated based on colony size measured once a week over a 2 week period. The sporulation ability of the wild-type strain XJ2008 and transformants were preceded by adding 200 μL of the conidial suspension of 1×106 spore mL-1 to 50 mL of liquid Czapek Dox medium, incubating the cultures at 25°C, 150 rpm for 7 days. A hemocytometer was employed to count the amount of the sporulation ability. Furthermore, the ability of microsclerotia formation was estimated through spreading 150 μL of the conidial suspension of 106 spore mL-1 on the Basal Modified Medium (BMM) covered with a sterilized permeable cellophane disc (HC273 film, Beijing Dongguo Co., China) as describe by Hu et al. (2013). The experiment was repeated three times and each replicate had three plates.
Pathogenicity test: Four-week-old cotton seedlings of cultivar Jimian11 (susceptible to Verticillium wilt) were used to evaluate the pathogenicity of V. dahliae strain XJ2008 and its mutants by rootdip-inoculation method with conidia suspension of 1×106 spore mL-1 as described by Hu et al. (2013). The wild-type strain XJ2008 and sterile distilled water served as positive and negative controls, respectively. Plants were then incubated in the green house under the control of temperature at 25°C. The symptoms of disease scoring were as follows: for, the severity of the inoculated plants was recorded at 3 weeks post-inoculation (wpi) following five categories: 0 = healthy plants, no symptoms on leaves, 1 = One or two cotyledonous leaves showing symptoms; 2 = A single true leaf showing symptoms, 3 = More than two leaves showing symptoms and 4 = dead plant. The Disease Index (DI) was calculated using the following formulate:
where, Ni is number of plants with a disease score of i
Data analysis: All data was analyzed using SPSS software (Version 16.0, SPSS Inc.). A one-way analysis of variance (ANOVA) and Student-Newman-Keuls (S-N-K) tests were employed to evaluate the statistical significance (p<0.05) of the results.
RESULTS
Optimized condition for ATMT: Experimental results showed that the growth of V. dahliae strain XJ2008 hyphae was completely inhibited at 50 g mL-1 hygromycin B with 200 g mL-1 cefotaxime sodium. Also, we found out that the transformation efficiency increased with rising concentrations of AS, which had obvious effects on transformation efficiency. The presence of AS (200 mol L-1) was the optimal concentration for V. dahliae transformation and yielded 89.75±5.40 transformants that identified per 106 conidia. However, fewer transformants were observed when the concentration of AS was increased (Fig. 1a). Agrobacterium tumefaciens concentration was increased from OD600 = 0.6-0.8 and obtained 69.25±1.65 and 80.00±3.13, respectively (Fig. 1b). In this study, optimal transformation frequency was attained when the mixture was co-cultivated for 36 h and obtained 82.00±1.47 transformants per 106 spore, while extended the incubation time could reduce transformation efficiency (Fig. 1c). The results indicated that the optimal temperature for achieving the highest transient transformation was 25°C with 82.75±3.94 (Fig. 1d). Strain AGL-1 yielded much more transformants than EHA105.
| Fig. 1(a-d): | Optimization of ATMT for wild-type strain XJ2008 of Verticillium dahliae, (a) Efficiency of acetosyringone (AS) on transformation was tested by adding to the IM plates with AS concentrations of 0, 100, 200, 300 and 400 μM, (b) Agrobacterium tumefaciens concentration. Two hundred microlitre of A. tumefaciens cell suspension (OD600 = 0.4, 0.6, 0.8, 1.0 and 1.2) was mixed with 200 of fresh spore suspension (106 spore mL-1) and then incubated on an IM agar plate containing 200 mol L-1 of AS at 25°C for 36 h. (c) Co-Culture time. Two hundred microlitre of A. tumefaciens cell suspension (OD600 = 0.8) was mixed with 200 of fresh spore suspension (106 spore mL-1) and then incubated on an IM agar plate containing 200 mol L-1 of AS at 25°C for 12, 24, 36 and 48 h (d) Co-Culture temperature. Two hundred microlitre of A. tumefaciens cell suspension (OD600 = 0.8) was mixed with 200 of fresh spore suspension (106 spore mL-1) and then incubated on an IM agar plate containing 200 mol L-1 of AS at 23, 25 and 28°C for 36 h. Each value indicates the mean and standard error |
Mutant library construction of defoliating strain XJ2008 and its evaluation: We got 4890 mutants from wild-type strain XJ2008 of V. dahliae using ATMT method. The transformants were identified using Hyg B resistance and Hyg-specific PCR analysis. A single expected band of about 1 kb was amplified from all of them using primers Hyg-F and Hyg-R with pCT-Hyg as positive control and the DNA from wild-type strain XJ2008 was used as the negative control (Fig. 2) and Southern blot analysis was performed from eleven PCR positive transformants with wild-type strain DNA as negative control and linearized pCT-Hyg as positive control.
| Fig. 2: | Transformant identification of Verticillium dahliae strain XJ2008 using hph gene specific primers Hyg-F and Hyg-R. Lane M: DNA ladder (D2000, Beijing, Tiangen Biotech Co.,China), Lanes 1-15: Genomic DNA isolated from putative transformantsVdAG2, VdAG6, VdAG7, VdAG9, VdAG11, VdAG16, VdAG18, VdAG20, VdAG23, VdAG25, VdAG26, VdAG27, VdAG28, VdAG30, VdAG33 and VdAG36, respectively, Lane 16: Genomic DNA of wild-type XJ2008, Lane 17: Vector pCT-Hyg as positive control |
| Fig. 3: | Southern blot analysis of T-DNA integration from eleven PCR positive transformants and a wild-type strain was digested with EcoRI. M DNA molecular weight markers with bases indicated on the left. Lanes 1-11: Transformants of strain XJ2008, Lane 12: Linearized pCT-Hyg as positive control and CK genomic DNA of wild-type XJ2008 |
The hybridization yielded only a single-copy insertion and the insertion sites are different for these 11 transformants. Moreover, the presence of different sized bands in the Southern blot revealed random integration of the T-DNA of the majority of the transformants. No hybridizing band was detected in the lane loaded with DNA from the untransformed wild-type strain (Fig. 3).
Mitotic stability of transformants is important feature of an effective mutagenesis system. After five subcultures in the absence of hygromycin B, V. dahliae transformants could still grow on PDA containing 50 μg mL-1 hygromycin B, suggesting that the hph gene was stably maintained in all the transformants.
The growth rate for wild-type strain was 6.75±0.18 mm per day. The 34.7% of mutants was significantly reduced the growth rate (p<0.05) andVdAG151had the lowest growth rate only it had 4.8±0.12 mm per day. Also 34.8% of mutants increased their growth rate, 16.8% lost their ability to produce microsclerotia when culturing on BMM for 30 days and 30% decreased their sporulation ability (Table 1). The 22.9% of mutants showed abnormal morphological characteristics including the deformation and fan-shaped colonies. There were 12.7% of mutants grew significantly colony edges that had more mycelia compared with the wild-type strain, aerial mycelia grew vigorously and the colony surface was swollen. The rest mutants had similar productivity to that of wild type strain XJ2008 that was folded (Table 1).
| Table 1: | Comparison of some important biological characteristics and pathogenicity between wild-type strain XJ2008 and its mutants |
dpi: Days post inoculation. The values in the table above were the average of three replicates for wild-type strain XJ2008 and its transformants, respectively and the value following by "±" was standard error. Values were significantly different (p<0.05) in S.N.K test of one-way ANOVA. | |
| Fig. 4(a-e): | Pathogenicity test of mutants using Jimian 11. Fourteen-day-old cotton seedlings were inoculated with conidia of wild-type or random T-DNA insertion mutants of Verticillium dahliae. The disease was scored at 21 days after inoculation based on a five categories: (a) Healthy plants, no symptoms on leaves, (b) One or two cotyledonous leaves showing symptoms, (c) A single true leaf showing symptoms, (d) More than two leaves showing symptoms and (e) Plant dead. The pathogenicity of mutant VdAG30, VdAG153, VdAG151 and VdAG95 was reduced relative to WT |
The pathogenicity test results showed that about 27.1% of mutants reduced their pathogenicity without development of symptoms, including VdAG30, VdAG151, VdAG153 and VdAG95 (Table 1 and Fig. 4). The other 57.0% of mutants caused similar symptoms to the wild-type and 15.9% of mutants were caused higher pathogenicity (Table 1).
DISCUSSION
Random mutagenesis through ATMT followed by inoculation of the generated mutants on host plants has been widely used to identify pathogenicity or virulence genes in several plant pathogens (Mullins et al., 2001; Jeon et al., 2007; Huser et al., 2009; Michielse et al., 2009; Munch et al., 2011; Giesbert et al., 2012). In present study, we optimized the conditions of ATMT that affect the transformation efficiency of inserted T-DNA in defoliating Strain of V. dahliae. Thereafter, a library consisted of 4890 mutants was constructed.
To carry out the transformation, we checked the optimal concentration of hygromycin B for wild type XJ2008. It was found that 50 μg mL-1 hygromycin B was completely inhibited the growth of wild type V. dahliae. This result suggests that this concentration can be used in selecting transformants and hygromycin B that is a suitable marker for V. dahliae.
The efficiency of transformation was 8-100 transformants per 106 spore, which was not very high compared to those of other fungi (De Groot et al., 1998; Hirabayashi et al., 1989; Mullins et al., 2001). The efficiency of fungal ATMT is affected by several factors, including: a) concentration of AS, b) A. tumefaciens strain and its growth conditions and (c) Numbers of fungal and bacterial cells were mixed for co-cultivation (Bundock et al., 1995; De Groot et al., 1998; Islam et al., 2012; Mullins et al., 2001; Rho et al., 2001). Several factors affecting the efficiency of transformation were tested and optimized in this study, such as influence of Agrobacterium strains, AS concentration; co-cultivation temperature; recipient Agrobacterium concentration and co-cultivation time were determined. This study facilitates strain improvement of defoliating Strain XJ2008 by molecular approaches and is a good reference for genetic study of other fungi.
AS was found to be necessary for A. tumefaciens mediated transformation, adjustments of AS concentration during the induction and co-cultivation stages may help to improve the transformation efficiency, which is also validated by our results in which an optimum amount of AS (200 mol L-1) is added. In contrast to previous reports AS in IM was dispensable for the growth of bacterial cells prior to co-cultivation (Michielse et al., 2005). AS is typically added to fungal transformations at a concentration of 200 mol L-1 (De Groot et al., 1998; Covert et al., 2001; Dos Reis et al., 2004). However, a high concentration of AS hindered the transformation, probably due to the toxic effects of high AS concentration posed on the recipient conidia, for example, inhibiting the germination of the spore.
The ratios of bacterial and fungal mixtures during co-cultivation may have a considerable effect on the transformation efficiency (Maruthachalam et al., 2008). In our study, Fig. 1b, the highest transformation frequency was observed with an A. tumefaciens density between OD600 = 0.6 and 0.8. However, the number of transformants decreased, when the concentration of A. tumefaciens reached to OD600 = 1.0 and 1.2. The increased concentration of A. tumefaciens may result in its overgrowth and lead to limit fungal growth, If the huge growth of A. tumefaciens also rises difficulty at the selection stage (Michielse et al., 2004; Yamada et al., 2009).
Up-till now, optimal temperatures were usually defined among 23, 25 and 28°C. Although, lower and higher values had been tested as well (Michielse et al., 2005). Our results demonstrated that higher transformation efficiency could be achieved at 25°C, the different efficiencies among these temperatures suggested that a closely low temperature during co-culture is beneficial for A. tumefaciens to transfer its T-DNA to the host (Combier et al., 2003; Gardiner et al., 2004; Michielse et al., 2004). Even though at 28°C hygromycin-resistant transformants also appeared (Fullner and Nester, 1996).
Generally, more transformants appeared by a longer co-cultivation period in most fungi (Yang and Lee, 2008; Zhang et al., 2008; Ando et al., 2009). In our study, we found that the transformation efficiency was improved by increasing the co-cultivation time up to 36 h. With the 36 h of co-cultivation the transformants were generated around 84 transformants per 1×106 spore mL-1 and increased time might lead to Agrobacterium background growth. The current study suggests that co-cultivations for 36 h appeared as the optimum time to obtain an adequate number of single copy of transformants.
The efficiency of two strains of A. tumefaciens namely, AGL-1 and EHA105 were tested for genetic transformation in this study. Agrobacterium strain AGL-1 presented the highest transformation rate at 54%, followed by EHA105 with 45%. Using the EHA105 strain, Cruz-Mendivil et al. (2011) have reported efficiencies of transformation up to 19%, whereas Sun et al. (2006) have reported transformation rates ranging from 4-36% with EHA105 and it is derived from super virulent strain A 281 (Hood et al., 1993). Studies which compare the relative transformation efficiencies of A. tumefaciens strains have demonstrated that strain AGL-1 is superior to C58, GV3101 and EHA105 in its ability to deliver T-DNA into Switch grass seedlings (Chen et al., 2010). Figure 2 showed that the PCR results revealed 1 kb of fragments were present in all five transformants, but not in the wild type strain. These results indicate that hygromycin B resistant colonies were obtained by ATMT system.
Mitotic stability of transformants is important feature of an effective mutagenesis system. After five subcultures in the absence of hygromycin B, V. dahliae transformants grew on PDA containing 50 μg mL-1 hygromycin B. The results suggested that the hph resistances were stably maintained in all the transformants.
Based on the above described results, we obtained 4890 mutants from wild-type strain XJ2008, Two hundred microlitre of fresh spore suspension (106 spore/mL) was mixed with an equal volume of A. tumefaciens cell suspension (OD600 = 0.8) and was spread onto cellophane placed on IM plates amended with 200 mol L-1 of AS. After Co-Cultivation at 25°C for 36 h, the cellophane was transferred onto a new PDA plates augmented with 200 g mL-1 of cefotaxime and 50 g mL-1 of hygromycin B and cultivated for 5-7 days to select transformants. Improved transformation efficiency was compared with that previously reported (Maruthachalam et al., 2011).
Screening of 196 mutants was distinct in providing virtually complete coverage of the value of the ATMT methodologies in investigating the molecular mechanisms that involved of defoliating V. dahliae and identification of genes directly or indirectly by characterizing the biological properties as well as pathogenicity of transformants.
Moreover, we report a high frequency of isolating pathogenicity/virulence-defective mutants compared with other fungi (Jeon et al., 2007; Talhinhas et al., 2008), because these mutants that were selected for pathogenicity assay showed interesting phenotypes on PDA medium, such as slow and fluffy growth, different colony color or deformation and fan-shaped colonies, decreased or increased the sporulation ability and lost the ability to produce microsclerotia. Among the identified genes through ATMT insertional mutagenesis, two had an insertion in genes that had previously been characterized and known to be involved in pathogenicity in other fungi and bacteria (De Lorenzo et al., 1997; Goswami and Kistler, 2004; Maruthachalam et al., 2011). In the determination of virulence and sporulation, the sporulation was reduced; however, it could be effective to spread the V. dahliae and would not have any difference in its virulence. This assay is preferred because it is rapidly compared with the conventional greenhouse assay in the soil medium for16 weeks (Klosterman and Hayes, 2006).
In addition, studying the impact on pathogenicity of V. dahliae is greatly important. We have developed two basic tools in order to study fungal interaction i.e., i) that is an efficient tool of ATMT to increase transformation and (ii) a transformation protocol to establish a mutant library in a virulent defoliating Strain XJ2008. These tools will provide a favorable reference for gene in the defoliating strain. Importantly, the V. dahliae defoliating strain is potentially useful for development-related genes in V. dahliae in the future.
ACKNOWLEDGMENTS
This research was supported by NSFC (31371888) and by 111 Project from Education Ministry of China (B07049).