Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum

ABSTRACT Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.     

抗氰烯菌酯假禾谷镰孢突变体的诱导及其生物学特性

为评估引起小麦茎基腐病的病原菌假禾谷镰孢Fusarium pseudograminearum对氰烯菌酯的抗性风险,对5株敏感菌株进行了室内药剂驯化,获得33株抗性突变体,突变频率为16.5%,其对氰烯菌酯的抗性水平范围为7.39～1 665.76倍,3株表现低抗,4株表现中抗,26株表现高抗;发现在myosin-5基因上存在11种抗性突变类型,其中217位的丝氨酸突变为亮氨酸(S217L)、420位的谷氨酸突变为赖氨酸(E420K)和135位的丙氨酸突变为苏氨酸(A135T)为主要突变类型,其比例分别为45.5%、15.2%和9.1%。S217L型抗性突变体的产孢量显著下降,菌丝生长速率和致病力与亲本菌株无显著差异。E420K型抗性突变体的菌丝生长速率和致病力显著下降,产孢量与亲本菌株无显著差异。A135T型抗性突变体的菌丝生长速率和产孢量与亲本菌株无显著差异。研究结果表明假禾谷镰孢在药剂选择压力下易形成氰烯菌酯的抗性群体,对氰烯菌酯存在中到高等的潜在抗性风险,其myosin-5的点突变与其对氰烯菌酯的抗性相关。     

胶孢炭疽菌CgSho1基因的克隆与功能分析

HOG-MAPK(high osmolarity glycerol mitogen-activated protein kinase)信号途径是真菌MAPK途径中参与渗透压响应的一条重要通路,在植物病原菌生长发育及致病过程中发挥着重要的作用。Sho1(synthetic high osmolarity-sensitive protein1)是HOG-M APK信号途径上游的一个重要感受器,在不同真菌中常具有不同的功能。本研究从胶孢炭疽菌中克隆了Sho1的同源基因,命名为Cg Sho1,该基因编码一个291个氨基酸的蛋白,含有4个跨膜结构域和一个SH3功能域。利用同源重组的方法获得了该基因的敲除突变体,与野生型相比,敲除突变体表现为营养生长缓慢,菌丝稀疏且疏水性增强,产孢量下降,对氧化压力和渗透压更加敏感,致病力明显减弱。上述结果表明,Cg Sho1参与调控胶胞炭疽菌的营养生长、分生孢子产量、氧化应激反应、渗透压响应及致病性。     

Detection and Visualization of the Major Acidic Exopolysaccharide of Ralstonia solanacearum and its Role in Tomato Root Infection and Vascular Colonization

Exopolysaccharides play an important role in the pathogenicity of Ralstonia solanacearum. We compared in vitro and in planta exopolysaccharide production of the pathogenic strain AW1 with that of three related mutant strains impaired in both their exopolysaccharide production and aggressiveness on tomato. The distinction between the two hexosamine-rich exopolysaccharides, namely the N-acetyl-glucosaminorhamnan and the major N-acetyl-galactosamine-containing acidic polymer was emphasized. The major acidic polymer was identified specifically by electron microscopy using glutaraldehyde/ruthenium red/uranyl acetate staining, by immunofluorescence using specific monoclonal antibodies and correlated to an appropriate biochemical analysis. The two mutant strains AW1-1 and AW-19A were totally devoid of any production of the major exopolysaccharide in vitro or in planta whatever the technique used. Infection and vascular colonization of tomato roots by the pathogenic strain were also compared to those of the mutant strains by light microscopy. Pathogenicity on tomato was assessed by root infection without any artificial injury. Light microscopy showed that the two mutant strains AW1-1 and AW-19A were poorly infective and unable to invade xylem vessels, while they induced defence mechanisms in root tissues and appeared aggregated or degenerated within cortical infection pockets. These two mutant strains were non-pathogenic or weakly aggressive, respectively. In contrast, the pathogenic strain AW1 and the hypoaggressive AW1-41 strains, which produce large amounts of the major acidic exopolysaccharide in planta, were both infective and invasive, and tomato root tissues exhibited only limited defence reactions. Thus, the major acidic exopolysaccharide produced by Ralstonia solanacearum is involved in root infection and vascular colonization, though its precise role is still unknown.     

Dothistromin toxin is not required for dothistroma needle blight in Pinus radiata

The pine needle blight pathogen Dothistroma septosporum produces a polyketide toxin, dothistromin. This paper reports that loss of the ability to produce dothistromin did not affect the pathogenicity of D. septosporum to Pinus radiata in a laboratory-based pathogenicity test. However, dothistromin synthesis provided an advantage to the D. septosporum wild-type, compared to dothistromin-deficient mutants, in growth competition with other fungi in vitro . Other pine-needle inhabitants, such as the latent pathogen Cyclaneusma minus and the endophyte Lophodermium conigenum , were inhibited by dothistromin-producing D. septosporum . Therefore, it was concluded that dothistromin is not a pathogenicity factor, but that it may play a role in competition of D. septosporum with other fungi in its ecological niche.     

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.     

草莓多主棒孢霉小柱孢酮脱水酶基因CcSCD1的功能研究

 多主棒孢霉(Corynespora cassiicola)是世界范围内重要的植物病原真菌,其引起的草莓棒孢霉叶斑病对草莓产业的健康发展具有潜在威胁。小柱孢酮脱水酶(scytalone dehydratase,SCD)是真菌多聚二羟萘类(DHN)黑色素生物合成途径中的关键酶,在植物病原菌致病过程中发挥重要作用。本研究通过同源重组的方法获得了草莓多主棒孢霉小柱孢酮脱水酶基因(CcSCD1)的敲除突变体,并进行了回补和RT-PCR验证。与野生型相比,敲除突变体△CcSCD1-2表现为菌落无色素沉积、菌丝稀疏、产孢量显著下降、分生孢子无色较小以及致病力明显减弱。结果表明,CcSCD1参与调控多主棒孢霉的黑色素生物合成、营养生长、分生孢子产量及致病力。     

Isolation of Pathogenicity Mutants of Fusarium oxysporum f. sp. melonis by Insertional Mutagenesis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to isolate mutants of Fusarium oxysporum f. sp. melonis impaired in pathogenicity. The race 2 strain Mel02010 was transformed with linearized pSH75, conferring resistance to hygromycin B, with or without the enzyme used to linearize the plasmid. Addition of restriction enzymes did not affect the transformation frequency. A total of 2929 REMI transformants were tested for pathogenicity to three melon cultivars, Amus, Ogon 9 and Ohi. The race 2 strains are pathogenic to Amus and Ogon 9, but not to Ohi. Of 43 transformants with reduced pathogenicity on susceptible melon cultivars, 12 mutants were examined in detail for pathogenicity, vegetative growth and integrative mode of pSH75. The levels of pathogenicity varied among these mutants. Two mutants (B48 and B137) almost completely lost pathogenicity to both susceptible cultivars, and the others had reduced pathogenicity. Mutants B48, B241, B886 and X36 were also impaired in vegetative growth. Mutant B809 was a biotin auxotroph. By DNA gel blot analysis, nine mutants were found to contain a single copy of the transformation vector. These mutants may thus be useful in isolating genes involved in pathogenicity. Received 22 December 2000/ Accepted in revised form 16 April 2001     

katExoo基因缺失突变对水稻白叶枯病菌H2O2抗性和致病性的影响

 为了揭示过氧化氢酶基因katExoo在水稻白叶枯病菌(Xanthomonasoryzaepv.oryzae,Xoo)过氧化氢(H₂O₂)抗性和致病性中的功能,本研究构建了基因缺失突变体ΔkatExoo,测定了突变体的H₂O₂抗性、过氧化氢酶(CAT)活性、在离体培养条件下的生长速率以及对水稻的致病性。用标记基因置换法获得了ΔkatExoo突变体,其保守的CAT结构域(GATase1_catalase和catalase_clade_2)被Gm^R片段所替换。katExoo基因缺失突变并不导致病菌的H₂O₂抗性和CAT活性降低或丧失,反而在一定程度上使之增强和升高。ΔkatExoo离体生长量显著降低,水稻接种叶片病斑明显缩短、在叶组织内的种群量下降。表明基因缺失突变显著地影响了病菌的生长、定殖和致病性。本研究结果为“KatExoo可能是Xoo的一个毒性因子”的假设提供了遗传学证据。     

Variability Resulting from Selfing and Outcrossing in Phytophthora cactorum

Self and hybrid oospore progeny and zoospore progeny of two pairs of Phytophthora cactorum mutants carrying a metalaxyl-resistance (M^r) nuclear gene or a chloroneb resistance (Cn^r) mitochondrial gene were compared for variation in growth rate, production of sporangia and oospores, and pathogenicity. Zoospore progeny were relatively uniform, whereas oospore progeny resulting from both selfing and outcrossing displayed great dissimilarity in all the traits tested. For pathogenicity, both pairs of tested mutants had greater variation in hybrid progeny than self progeny. For growth rate and oospore production, the same is true only for the first pair, but not the second pair. The variation in sporangium production displayed by one parental self progeny was greater than that by hybrid progeny in both pairs tested. These results show that isolates and traits selected for study have considerable effect on the variation in hybrid and self progeny and suggest that different mechanisms may be operating in creating variation in these two types of progeny. Received 23 October 2000/ Accepted in revised form 11 January 2001     

苹果树腐烂病菌转录因子VmSte12的鉴定及功能分析

 Ste12是最早在酿酒酵母(Saccharomyces cerevisiae)中发现的一类转录因子。之后在部分丝状真菌中也发现了该类转录因子的存在,并证明其能够参与调控生殖生长以及病原真菌的致病力。然而,苹果树腐烂病菌(Valsa mali)中是否存在该类转录因子以及其功能尚不明确。本研究基于生物信息学鉴定苹果树腐烂病菌中Ste12的同源基因,采用烟草瞬时表达系统进行蛋白亚细胞定位分析,利用酵母单杂技术进行转录激活功能分析,进而利用PEG介导的原生质体转化技术构建基因缺失突变体,并对其进行表型观察与分析。结果表明,苹果树腐烂病菌存在Ste12的同源基因,我们将其命名为VmSte12。该基因编码701个氨基酸,包含Ste同源结构域和C₂H₂锌指结构域。VmSte12定位于细胞核,并具有体外转录激活功能。与野生型相比,VmSte12敲除突变体生长速率平均下降10.1%,分生孢子器数量平均下降94.6%,致病力平均下降27.4%。可见,VmSte12具有Ste12类转录因子特征,并且极有可能在腐烂病菌的生长、繁殖、致病等方面发挥重要作用。     

Pathogenicity of Streptomyces scabies Mutants Altered in Thaxtomin A Production

ABSTRACT To investigate the role of thaxtomin A in the pathogenicity of Streptomyces scabies, mutants altered in thaxtomin A production were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutants of S. scabies EF-35 could be differentiated according to levels of thaxtomin production. Mutants M1, M8, and M19 produced 2 to 20 times less thaxtomin A in oat bran medium than did EF-35. M1 and M19 were deficient in tryptophan catabolism. Thaxtomin production was reduced by about 300 times in mutant M16, which was a glutamic acid auxotroph. No thaxtomin A was detected in M13 culture supernatant. This mutant had a normal growth rate, was prototrophic, and catabolized tryptophan. Pathogenicity of mutants was tested on radish and potato. Mutants M1, M8, and M19 were pathogenic but, in most cases, less virulent than EF-35. M13 and M16 were nonpathogenic. These results suggest that thaxtomin A is an important pathogenicity determinant in S. scabies.     

Phage sensitivity in relation to pathogenicity and virulence of the cotton bacterial blight pathogen of Sudan

At least two pathotypes of Xanthomonas campestris pv. malvacearum are now known to exist in Sudan. The pre-Barakat (race 1) and post-Barakat (race 2) pathogens have been shown to exhibit different host specificity. The former is pathogenic and highly aggressive on only cultivars with no resistance genes or with the B₂ and/or B₃ resistance factors, while the latter can infect the B₆ cultivars also. Race 2 in Sudan, which was previously reported to infect all the standard differentials, produced milder angular leaf spot symptoms and occasionally restricted vein infection. Moreover, it exhibited reduced growth in planta compared with race 1.
Bacteriophage studies revealed that the two races are quite distinct in their phage sensitivity. Race 1 can be lysed by only three, or rarely four, of the six phages used for typing, while race 2 is sensitive to all of them. The present study suggests that phage 7 may be the type-determining phage for race 2. Race 2 strain mutants resistant to phage 3 or 4 were found to be sensitive to phage 7 and pathogenic to both Acala and Barakat, although showing marked attenuation of virulence. However, mutants resistant to phage 2 or 7 were insensitive to all the phages and although they retained their pathogenicity to Acala, they either lost the ability to infect Barakat or produced a hypersensitive reaction. The resistance of all mutants was found to be due to failure to adsorb the homologous phage, indicating a change in the cell wall. The association of this with the attenuation of virulence suggests that bacterial wall components may function as virulence determinants in Xanthomonas campestris pv. malvacearum.     

尖孢镰刀菌番茄专化型中SNARE蛋白FolSso1的功能分析

 SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor)蛋白保守存在于丝状真菌中,在膜泡转运的过程中起着关键的作用。番茄枯萎病是由尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,Fol)引起的,严重威胁着番茄的生产。我们使用反向遗传学的方法来研究番茄枯萎病菌中SNARE蛋白FolSso1的功能,实验结果发现FolSSO1的基因缺失突变体菌丝生长速率降低,且产孢数量减少。另外,FolSSO1基因的缺失导致突变体相较于野生型菌株对细胞壁压力与细胞膜压力更加敏感。然后,在番茄果实和番茄植株的致病性实验中,我们发现FolSSO1的缺失并没有引起Fol致病性显著的变化。综上所述,本研究发现FolSSO1可以调控Fol营养生长,繁殖和对环境压力的响应过程,然而对Fol的致病过程并没有显著的调控作用。     

Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

ABSTRACT Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was approximately 46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance.     

尖孢镰刀菌番茄专化型中SNARE蛋白FolSso1的功能分析

 SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor)蛋白保守存在于丝状真菌中,在膜泡转运的过程中起着关键的作用。番茄枯萎病是由尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,Fol)引起的,严重威胁着番茄的生产。我们使用反向遗传学的方法来研究番茄枯萎病菌中SNARE蛋白FolSso1的功能,实验结果发现FolSSO1的基因缺失突变体菌丝生长速率降低,且产孢数量减少。另外,FolSSO1基因的缺失导致突变体相较于野生型菌株对细胞壁压力与细胞膜压力更加敏感。然后,在番茄果实和番茄植株的致病性实验中,我们发现FolSSO1的缺失并没有引起Fol致病性显著的变化。综上所述,本研究发现FolSSO1可以调控Fol营养生长,繁殖和对环境压力的响应过程,然而对Fol的致病过程并没有显著的调控作用。     

Characterization and functional analysis of clpB gene from Acidovorax avenae subsp. avenae RS‐1

The type VI secretion system (T6SS) has been reported to be highly associated with various cellular activities in strain RS‐1 of Acidovorax avenae subsp. avenae (Aaa), the pathogen of bacterial brown stripe of rice. However, the role of the clpB gene that presents in the T6SS gene cluster in Aaa pathogenicity has not been clarified. The aim of the current study was to characterize the function of clpB and to investigate its contribution to bacterial pathogenesis using insertional deletion mutation and complementation approaches. The results indicated that mutation of clpB significantly affected bacterial growth, virulence, exopolysaccharide (EPS) production, biofilm formation and expression of 13 other T6SS genes of Aaa RS‐1. The reduction of virulence may be also partially due to the change in EPS composition, which was characterized by the Fourier transform infrared (FTIR) spectra. Furthermore, analysis of protein homology modelling showed that the structure of ClpB is different from those of the other T6SS components. In addition, structural difference was observed between ClpB and Type IV pili (TFP) as well as Type IV pilus biogenesis proteins (PilP), whose functions are similar to ClpB. Taken together, this study demonstrated that the clpB gene plays a key role in Aaa bacterial virulence.     

Monomorphic Nonpathogenic Mutants of Ustilago maydis

ABSTRACT We have developed conditions which promote the dimorphic transition of haploid cells of Ustilago maydis in vitro by controlling the pH of the media. At low pH (below 5.0) mycelial growth occurs, whereas at neutral pH yeastlike growth takes place. We screened for mutants unable to form mycelium at low pH and obtained 26 mutants. These mutants have been characterized by their cell and colony morphology in different media. Mutations in 18 strains were found to be recessive when these strains were crossed with the wild type. Other crosses indicated that they were affected in genes other than a and b. Crosses between mutants suggest that the mutations fall in at least two complementation groups. In addition, mutants were characterized by their pathogenicity to corn seedlings. Mutations which were recessive for pathogenicity were also recessive for morphogenesis in vitro.     

禾谷镰刀菌脂质转运蛋白Vps13的功能分析

 Vps13蛋白家族是真核生物中高度保守的一类脂质转运蛋白,其在丝状真菌中的功能尚不清楚。小麦赤霉病主要由禾谷镰刀菌(Fusarium graminearum)引起,是小麦最重要病害之一。禾谷镰刀菌含有酿酒酵母VPS13的一个同源基因FgVPS13。通过同源重组的方法获得禾谷镰刀菌FgVPS13敲除突变体。研究表明,FgVPS13敲除突变体出现生长、产孢和有性生殖的缺陷。FgVPS13敲除突变体在小麦胚芽鞘和麦穗上的致病力下降,产生的脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)毒素含量也明显降低。而且,FgVPS13抑制线粒体自噬。总之,FgVPS13参与调控禾谷镰刀菌菌丝生长、无性和有性生殖、致病力和线粒体自噬。     

含hrp基因的克隆片段在水稻白叶枯病菌不同小种中的功能和同源性

 将从水稻白叶枯病菌(Xanthomonas campestris pv.oryzae Dye,简称Xco)小种3菌株JXOⅢ中克隆的hrp基因片段(pNAX3103),用三亲交配法向病菌其它小种及其毒性基因突变体转移,结果表明pNAX3103可以进入其它小种,但对不同小种毒性基因突变体的转移能力则不同。功能互补分析表明;该hrp基因片段在不同小种中对病菌的生长,以及蛋白酶、果胶酶和纤维素酶的产生和分泌无明显影响;在野生型菌株中能增加亲本菌株对水稻品种IR26的致病力(JXO I)或亲和性(JXO V),对于毒性基因突变体,pNAX3103虽能进入JXO I经Tn5诱变的毒性基因突变体XcoM1107,但不能恢复其致病性和淀粉酶阴性反应。用hrp基因片段作为探针,对10个来源不同的小种或菌系群进行DNA同源性分析,结果与所有Xco菌株都有明显的同源性杂交带,但杂交带型不同,与甘蓝黑腐病菌、水稻细菌性条斑病菌、柑桔溃疡病菌、大白菜。软腐病菌的DNA有同源杂交带,但与水稻基腐病菌DNA无同源性。