Rhizosphere bacterial community in watermelon-wheat intercropping was more stable than in watermelon monoculture system under Fusarium oxysporum f. sp. niveum invasion

Plant and Soil - Microbial turnover processes are typically restricted by low substrate availability in the subsoil. We hypothesized that SOM decomposition increases with plant density and...     

Allelopathic effects of root exudates from watermelon and rice plants on Fusarium oxysporum f.sp. niveum

Root exudates have a key role in communication between plants and microbes in the rhizosphere. Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (Fusarium oxysporum), drastically reduces watermelon yields in continuous cultivation systems, but it can be significantly alleviated using watermelon/aerobic rice intercropping system as shown by the research carried out in this laboratory. It is important to evaluate the interaction between root exudates from the two crops and the pathogen and thus to clarify the mechanism of disease suppressiveness in the intercropping system. The effects of phenolic acids, sugars and free amino acids in root exudates from watermelon (REW) and rice (RER) on the growth of Fusarium oxysporum were studied. The results obtained are listed as follows: (1) REW significantly increased spore germination and sporulation, whereas RER had inhibitory effects on those two parameters. (2) HPLC analysis showed that salicylic acid, p-hydroxybenzoic acid and phthalic acid were identified in exudates from both plants, but p-coumaric acid was only detected in rice and ferulic acid only in watermelon. Moreover, of the total rice exudates a high proportion (37.9 %) of p-coumaric acid was detected and the total amount of phenolic acids was 1.4-fold as high as that in watermelon. (3) Considerable differences in the components and contents of both sugars and amino acids were found between REW and RER exudates. (4) Exogenously applied alanine (Ala) increased spore germination and sporulation. In contrast, addition of exogenous p-coumaric acid reduced spore germination and sporulation, relative to controls. It was concluded that the rice root exudates had anti-fungal properties while that from watermelon promoted pathogen growth. This discovery provided a scientific basis for practicing watermelon/aerobic rice intercropping to control Fusarium wilt in watermelon.     

西瓜枯萎病菌的快速检测与鉴定

通过西瓜枯萎病菌与其他专化型枯萎病菌及瓜类几种重要病原菌的比较基因组分析,获得了西瓜枯萎病菌的基因组特异序列。在此基础上,设计出特异引物,筛选可扩增出西瓜枯萎病菌特异性DNA条带的引物。将特异性引物和尖孢镰刀菌专化型的通用引物W106R/W106S结合,建立双重PCR检测体系。该双重PCR检测体系可以在一次PCR反应中快速、准确的检测出西瓜枯萎病菌,为通过分子方法快速鉴定西瓜枯萎病菌提供技术支持。     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil

We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp.niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Paenibacillus polymyxa SQR-21 systemically affects root exudates of watermelon to decrease the conidial germination of Fusarium oxysporum f.sp. niveum

Paenibacillus polymyxa SQR-21 has been identified as a potential agent for the biocontrol of Fusarium wilt in watermelon, which is caused by the pathogenic fungus Fusarium oxysporum f.sp. niveum (FON). In the present study, the effects of root exudates from watermelon plants inoculated or non-inoculated with either SQR-21 or FON on conidial germination of FON were investigated. Compared to the control, conidial germination was decreased with root exudates from SQR-21-inoculated plants, but conidial germination was enhanced by root exudates from FON-inoculated plants. Maximal germination was found with root exudates from FON-inoculated plants after 30 d, which was 1.35 times more germination than the control. A split-root system was designed to verify that the alterations of the exudation pattern in SQR-21- inoculated or FON-inoculated watermelon roots were not only local, but also systemic. Cinnamic acid was found in the watermelon root exudates. An assay to test the effects of cinnamic acid on conidial germination of FON revealed that the stimulation of conidial germination was observed from cinnamic acid concentrations ranging from 0 to 30 μg/ml. In conclusion, both of SQR-21 and FON systemically affects watermelon root exudates. These results will help to the better understanding of the plant-microbe communication and will guide to improve the biocontrol strategies against Fusarium wilt of watermelon plants.     

In Vitro Physiological Responses of Fusarium oxysporum f. sp. niveum to Exogenously Applied Syringic Acid

ABSTRACT. Plant–microbe interactions are often accompanied by allelochemicals, such as syringic acid, released from the host plant. To explore the role of phenolic acids released from crop host plants in response to pathogen invasion, we examined the allelopathic effect of an artificially applied syringic acid on Fusarium oxysporum f. sp. niveum . We demonstrated that the growth and the conidial germination rate of F. oxysporum f. sp. niveum were stimulated at lower concentrations of syringic acid, though inhibited by higher dosage compared with control. The yield of fungus mycotoxin was increased from 60.9% to 561.5%. We conclude that syringic acid can be considered as a allelochemical inducer, stimulating the relative virulence factors of invading pathogens.     

Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato

We demonstrated that exogenous application of 200 μM salicylic acid through root feeding and foliar spray could induce resistance against Fusarium oxysporum f. sp. Lycopersici (Fol) in tomato. Endogenous accumulation of free salicylic acid in tomato roots was detected by HPLC and identification was confirmed by LC–MS/MS analysis. At 168 h of salicylic acid treatment through roots, the endogenous salicylic acid level in the roots increased to 1477 ng g^?1 FW which was 10 times higher than control plants. Similarly, the salicylic acid content was 1001 ng g^?1 FW at 168 h of treatment by foliar spray, which was 8.7 times higher than control plants. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidase (POD, EC 1.11.1.7) were 5.9 and 4.7 times higher, respectively than the control plants at 168 h of salicylic acid feeding through the roots. The increase in PAL and POD activities was 3.7 and 3.3 times higher, respectively at 168 h of salicylic acid treatments through foliar spray than control plants. The salicylic acid-treated tomato plants challenged with Fol exhibited significantly reduced vascular browning and leaf yellowing wilting. The mycelial growth of Fol was not significantly affected by salicylic acid. Significant increase in basal level of salicylic acid in noninoculated plants indicated that tomato root system might have the capacity to assimilate and distribute salicylic acid throughout the plant. The results indicated that the induced resistance observed in tomato against Fol might be a case of salicylic acid-dependent systemic acquired resistance.     

Detection of RAPD polymorphisms inGladiolus cultivars with differing sensitivities toFusarium oxysporum f. sp.gladioli

Fusarium oxysporum Schlecht. Fr. F. sp.gladioli (FOG) is the most important gladiolus pathogen. One of the most environmentally friendly methods to control its spread is to use cultivars that are minimally sensitive to the pathogen. Infected corm tissues in more resistant varieties have been shown to produce suberin layers that inhibit fungal iphae growth. RAPD analysis of genomes from 9 selected gladiolus cvs, chosen to be the most resistant and sensitive to FOG, were performed to verify DNA polymorphism levels. Total nucleic acid extraction was carried out with a chloroform-phenol method from tissues of plants in 3 growth stages. RAPD experiments were performed using 14 primers with varyingTaq polymerases and primer concentrations. Five of the primers tested gave no polymorphic profiles. Five primers produced polymorphic bands, allowing us to obtain RAPD profiles typical for one or more of the more resistant cvs. All the tested growth stages provided repeatable results, indicating the reliability of detected polymorphisms. Cloning the more interesting polymorphic DNA fragments in the future will verify the presence of specific genes related to FOG resistance mechanisms in gladiolus.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

Differential interaction between melon cultivars and race 1.2 of Fusarium oxysporum f.sp. melonis

One hundred and ten melon accessions from different origins were evaluated for resistance to race 1.2 of Fusarium oxysporum f.sp. melonis (Fom). Artificial inoculations were performed and plants were scored for presence or absence of symptoms. The screening revealed that sources of resistance to this fungus are limited. However, a few resistant accessions were found. The accessions 'Kogane Nashi Makuwa', 'C-211' and 'C-40' from Japan provided the highest resistance to both patotypes of race 1.2 of Fom which seems to confirm that the Far East is a rich area in resistances to this race. We also detect an interesting degree of resistance in 'BG-5384', a Portuguese accession that may be classified as belonging to the botanical variety inodorus.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

Identification of resistance against Fusarium oxysporum f.sp. lini in linseed germplasm

Screening of germplasm/varieties was made to find out the sources of resistance against F. oxysporum f. sp. lini. Screening was conducted on 78 available germplasm/varieties during 2003–2004 and 2004–2005 in rabi season of linseed under natural conditions. Out of total 78 entries, 27 cultures were found to be resistant to disease as the disease incidence in these cultivars were between 0 and 10%. Twenty-three cultivars fell in moderately resistant category with 10.1–25% wilt incidence. Nine genotypes were found moderately susceptible sho'wing 25.1–50% disease incidence, 14 genotypes were found susceptible showing 50.1–75% and 6 genotypes were found highly susceptible to disease (above 75%).     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

Wild Narcissus species as a source of resistance to Fusarium oxysporum f.sp. narcissi

Thirty three species and varieties of Narcissus plus seventeen cultivars with recent species parentage were screened for resistance to Fusarium oxysporum f.sp. narcissi. Accessions were either wholly resistant (bulbs remaining uninfected), partially resistant (a variable portion of the bulbs infected) or susceptible (all of the bulbs infected). Although a pattern of resistance was found among the species tested the genetic basis of the resistance is not known.