Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

Genetic Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Population structure and linkage disequilibrium in a large collection of Fusarium oxysporum strains analysed through iPBS markers

In the current study, 160 pathogenic strains of Fusarium oxysporum collected from tomato, eggplant and pepper were studied. Eighteen inter‐primer binding site (iPBS)‐retrotransposon primers were used, and these primers generated 205 scorable polymorphic bands. The number of polymorphic bands per primer varied between 9 and 19, with a mean of 11 bands per primer. The highest polymorphism information content (PIC) value was determined as 0.27, and the lowest was 0.05. The unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram including a heat map revealed that the 160 pathogenic strains of F. oxysporum were divided into two main clusters. The first cluster mainly included F. oxysporum f. sp. capsici (FOC) and F. oxysporum f. sp. melongenae (FOMG) isolates. The second cluster mainly comprised F. oxysporum f. sp. lycopersici (FOL) and F. oxysporum f. sp. radicis lycopersici (FORL) isolates. The highest percentage of loci in significant linkage disequilibrium (LD) was detected for FOL, whereas the lowest level of LD was found for FOC, and 95.2%, 99.4%, 99.1% and 97.4% of the relative kinship estimates were less than 0.4 for FOL, FOMG, FORL and FOC, respectively. LD differences were detected among formae speciales, and LD was higher in FOL as compare to FOC species. The findings of this study confirm that iPBS‐retrotransposon markers are highly polymorphic at the intraspecific level in Fusarium spp.     

Sciarid and shore flies as aerial vectors of Fusarium oxysporum f. sp. cucumerinum in greenhouse cucumbers

The options for managing Fusarium wilt in greenhouse cucumbers are limited by our poor understanding of the modes of survival and dissemination of the pathogen. This study uses a specific quantitative real‐time PCR assay for Fusarium oxysporum f. sp. cucumerinum to investigate the significance of flying insects as aerial vectors of the pathogen in a commercial cucumber greenhouse. Shore flies were more frequently detected (35.5%) carrying F. oxysporum f. sp. cucumerinum than sciarids (25%), with both species carrying between 1 × 10² and 1 × 10⁶ pathogen genome copies/individual. Sciarid and shore flies acquired F. oxysporum f. sp. cucumerinum following exposures to agar cultures of the pathogen of up to 94 h. Light microscopy revealed that spores were carried externally on the bodies of the adult flies. The ability of adult sciarid flies to vector the pathogen from peat‐grown diseased cucumber plants and infect healthy cucumber plants was demonstrated in a caged glasshouse trial. An inoculum density trial showed that vascular wilt disease was initiated after inoculation of peat‐grown seedlings with as few as 1000 conidia. We conclude that sciarid and shore flies play significant roles as vectors of F. oxysporum f. sp. cucumerinum in greenhouse cucumbers and need to be recognized in developing integrated crop management strategies.     

Effects of the biocontrol agent Bacillus amyloliquefaciens SN16‐1 on the rhizosphere bacterial community and growth of tomato

Fusarium oxysporum is a common soil‐borne pathogen that causes serious economic losses in tomato crops worldwide. The purpose of this study was to evaluate the influence of the bio‐control agents Bacillus amyloliquefaciens SN16‐1 and Pseudomonas fluorescens SN15‐2 and the pathogen Fusarium oxysporum f.sp. lycopersici (FOL) inoculation on tomato rhizosphere bacterial communities and growth, as measured by terminal restriction fragment length polymorphism (T‐RFLP). Treatment with SN16‐1 and SN15‐2 had a transient influence on indigenous bacterial communities, withSN16‐1 showing great potential for controlling FOL. The corresponding genera of terminal restriction fragments (T‐RFs) that were significantly altered after 10 days were obtained using Ribosomal Database Project (RDP) database comparison. Genera that produce antibiotics and promote plant growth were activated by SN16‐1 and FOL treatments, indicating that SN16‐1 responds quickly to FOL invasion. Moreover, the bioremediation activity characteristic of certain genera and the levels of enzymes that degrade pathogen cell walls were decreased while bacterial nutrient cycling and plant growth promotion were enhanced with FOL treatment. In conclusion, we found that SN16‐1 possesses the capacity to control tomato wilt, acts synergistically with soil microbes and does not have a persistent effect on the rhizosphere bacterial communities of tomato.     

Biological control of Fusarium wilt of tomato with <Emphasis Type="Italic">Fusarium equiseti</Emphasis> GF191 in both rock wool and soil systems

The plant growth-promoting fungus (PGPF) Fusarium equiseti GF191 was tested for its ability to control Fusarium wilt of tomato (FWT) caused by Fusarium oxysporum f. sp. lycopersici (FOL) in both a hydroponic rock wool and soil system. F. equiseti effectively controlled FWT, with protective effects based on disease severity of 66.7–88.6% in four experiments. The numbers of colony-forming units of FOL per gram fresh weight of stems were significantly reduced (P < 0.05) in plants treated with F. equiseti. Stem extracts from F. equiseti-treated and pathogen-challenged plants significantly inhibited the germination and germ-tube length of FOL microconidia and the production of FOL budding-cells. Tomatine content in tomato stems treated with F. equiseti was significantly increased compared with the non-treated control.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Quick and accurate detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">carthami</Emphasis> in host tissue and soil using conventional and real-time PCR assay

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40–80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.     

First Report of Stem Canker on Phoenix Tree (Firmiana simplex) Caused by Fusarium oxysporum in China

A stem canker disease was observed on the phoenix trees located in the region of Dezhou, Shandong province. Symptomatic stems were collected and evaluated for the possible casual agent of the disease. A fungus resembling Fusarium sp. was consistently isolated from pieces of symptomatic tissues. The fungus formed abundant aerial mycelium on potato dextrose agar and produced the micro‐ and macro‐conidia on carnation leaf agar. The nucleotide sequences of the internal transcribed spacer of the rDNA from three representative isolates showed 100% identical to those of Fusarium oxysporum isolates deposited in the GenBank database. On the basis of morphological characteristics, pathogenicity test and molecular identification, the causal agent was identified as F. oxysporum. To our knowledge, this is the first report of stem canker on phoenix tree caused by F. oxysporum in China.     

Development of a Loop‐Mediated Isothermal Amplification Assay to Detect Fusarium oxysporum

We report the development of a loop‐mediated isothermal amplification (LAMP) assay targeting the CYP51C element for visual detection of F. oxysporum which caused Fusarium wilt in soybean. The CYP51C‐LAMP assay efficiently amplified the target gene in 60 min at 62°C. And specificity was evaluated against F. oxysporum, Fusarium spp. and other fungal species. The detection limit of the CYP51C‐specific LAMP assay for F. oxysporum was four conidia per gram soil. The assay also detected F. oxysporum from inoculated soybean tissues and residues. These results suggest that this CYP51C‐LAMP assay can be used to detect residues on plants in the field.     

Induced resistance to Botrytis cinerea in Capsicum annuum by a Fusarium crude elicitor fraction,free of proteins

Fusarium oxysporum f. sp. lycopersici (FOL) induces resistance in pepper against the airborne pathogen Botrytis cinerea and the soil‐borne pathogen Verticillium dahliae. However, its practical use is limited due to its pathogenicity to other crops. In this study we tested several fractions of a heat‐sterilised crude FOL‐elicitor preparation to protect pepper against B. cinerea and V. dahliae. Only the protein‐free insoluble fraction of the preparation reduced B. cinerea infection. However, none of the fractions reduce V. dahliae symptoms. The insoluble protein‐free fraction induced expression of defence genes in the plant, namely a chitinase (CACHI2), a peroxidase (CAPO1), a sesquiterpene cyclase (CASC1) and a basic PR1 (CABPR1). Even though the CASC1 gene was not induced directly after treatment with the insoluble fraction in the leaves, it was induced after B. cinerea inoculation, showing a priming effect. The insoluble protein‐free FOL‐elicitor protected pepper against the airborne pathogen through a mechanism that involves induced responses in the plant, but different to the living FOL.     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato

Nine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37?mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

Differential Colonization Patterns of Bananas (Musa spp.) by Physiological Race 1 and Race 4 Isolates of Fusarium oxysporum f.sp. cubense

Fusarium oxysporum f.sp. cubense (Foc) is the causative agent of Fusarium wilt of bananas (Musa spp.). To clarify the colonization patterns of Foc in bananas, two green fluorescent protein‐tagged isolates, NT320 (race 1) and B2‐gfp (race 4), were used to follow infection of the banana varieties Pisang Awak and Brazil. Penetration and colonization of both isolates in roots of these two banana varieties were observed within 6 days, but sporulation in xylem vessels was not observed until day 30 postinoculation. Interestingly, B2‐gfp penetrated into xylem vessels of Pisang Awak banana roots more quickly than NT320, implying that the race 4 isolate is more virulent than the race 1 isolate. This result was further confirmed by comparing the disease severity of plants inoculated with NT320 with that of plants inoculated with B2‐gfp. Quantitative real‐time PCR revealed that some pathogenicity‐associated genes, including Fga1, Fhk1, Fow2 and Ste12, were upregulated by B2‐gfp during exposure to Brazil bananas, while they were either downregulated by NT320 or not significantly changed. These data might partly explain why the race 4 isolate was more virulent than the race 1 isolate.     

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.     

Presence of Fusarium oxysporum f. sp. melonis Race 1 in Soils Cultivated with Melon in the State of Colima (Mexico)

During years 2001, 2002 and 2003 the gravity of the Fusarium wilt in 1000 hectares of melon culture was evaluated in Colima (Mexico). In spite of the soil disinfections with methyl bromide, the losses could reach 25% of the final production. The analysis of 4 soil samples from the fields with ill plants, in a selective medium for Fusarium, allowed to detect the presence of F. oxysporum. By means of the presented technique “soil phytopathometry”, 31 isolates of F. oxysporum f. sp. melonis were obtained from the soil samples. The isolates were inoculated on melon plants to evaluate their pathogenicity. The 31 isolates inoculated, produced the symptoms of chlorosis and wilting, in melon cultivars that allowed us to affirm that all isolates were race 1 of F. oxysporum f. sp. melonis. Being this the first news of the presence of F. oxysporum f. sp. melonis in the state of Colima (Mexico).     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.