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.     

Contamination of Bananas with Beauvericin and Fusaric Acid Produced by Fusarium oxysporum f. sp. cubense

Background

Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis.

Methodology/Principal Findings

Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak ‘Guangfen #1’ and 10 Cavendish ‘Brazilian’ plants. Fusaric acid and BEA were detected in all the tissues, including the fruits.

Conclusions/Signficance

The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.     

Genetic Variability of Fusarium oxysporum f.sp. ciceris Population Affecting Chickpea in the Sudan

Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) is the most important soilborne disease of chickpea in the Sudan and many other countries. A total of 76 Foc isolates from six different chickpea‐growing states in the Sudan have been collected in this study to investigate the genetic diversity of Sudanese Foc isolates. Additional 14 Foc isolates from Syria and Lebanon were included in this study. All isolates were characterized using four random amplified polymorphic DNA (RAPD), three simple sequence repeats (SSR), five sequence‐characterized amplified region (SCAR) primers and three specific Foc genome primers. Based on the similarity coefficient, the results indicated two major clusters included seven subclusters. The isolates from the Sudan were grouped as identified as races 0, 2 and unknown races. The isolates from Syria and Lebanon were grouped together as they identified as races 1B/C and 6, respectively. This study identified a new race Foc (race 0) in the Sudan. The results of this study will be useful for breeders to design effective resistance breeding program in chickpea in the Sudan.     

Control of chickpea wilt (Fusarium oxysporum f.sp. ciceris) using Trichoderma spp. in Ethiopia

Thirty-two Trichoderma isolates were collected from soils grown with chickpea in central highlands of Ethiopia. The eight isolates were identified by CAB-International as Trichoderma harzianum, T. koningii and T. pseudokoningii. In in vitro tests, all Trichoderma isolates showed significant (P < 0.05) differences in their colony growth and in inhibiting the colony growth of Fusarium oxysporum f.sp. ciceris, race 3. In potted experiment, four Trichoderma isolates were tested as seed treatment on three chickpea cultivars (JG-62 susceptible, Shasho moderately susceptible and JG-74 resistant) against F. oxysporum f.sp. ciceris, race 3. The result showed that T. harzianum and unidentified Trichoderma isolate T23 significantly reduced wilt severity and delayed disease onset. The degree of wilt severity and delay of disease onset varied with chickpea cultivars. Our study revealed that biological control agents such as Trichoderma can be a useful component of integrated chickpea Fusarium wilt management.     

Artificial inoculation of banana tissue culture plantlets with indigenous endophytes originally derived from native banana plants

Fusarium wilt disease of banana is one of the most harmful fungal diseases affecting banana production worldwide. We hypothetically proposed that the loss of indigenous endophytes in tissue culture propagation of banana might be related to increased disease severity on banana plants. In the present study, a mixture of uncultivated endophytes, which was originally derived from native healthy banana plant in plantation, was used to artificially inoculate banana tissue culture plantlets. A broad spectrum of bacterial communities was detected in the roots of artificially inoculated plantlets by 16S ribosomal RNA gene analysis, and γ-Proteobacteria was identified as the dominant group. Banana wilt pathogen Fusarium oxysporum f. sp. cubense race 4 was inoculated to the plantlets after potting to investigate disease progress. With early diagnosis of fungal pathogen infection, 54% reduction was detected in artificially inoculated plantlets compared to endophyte-free control plantlets. The re-introduction of naturally-occurring endophytes into tissue culture banana plantlets led to a 67% suppression rate of wilt disease at the fifth month after pathogen infection on plantlets in the greenhouse. In addition to disease suppression, growth of host plantlets was also promoted with the inoculation of endophytes. The artificial inoculation method provided a foundational understanding of ecological enrichment to control banana wilt disease in future.     

Endophytic microorganisms as potential growth promoters of banana

The potential of endophytic microorganisms in promoting the growth of their host plant was determined by artificially introducing five isolates (bacterial and fungal strains: UPM31F4, UPM31P1, UPM14B1, UPM13B8, UPM39B3) isolated from the roots of wild bananas into both healthy and diseased banana plantlets (Berangan cv. Intan). The response of the host plants to endophytic infection was assessed by measuring the change in four growth parameters: plant height, pseudostem diameter, root mass and total number of leaves. The endophytes tested as growth promoters were found to have a significant effect in both healthy and Fusarium-infected (diseased) plantlets. In both experimental systems, the bacterial isolate UPM39B3 (Serratia) and fungal isolate UPM31P1 (Fusarium oxysporum) showed promising growth-promoting properties. Isolate UPM39B3 (Serratia) induced the largest increases in all four growth parameters in healthy plantlets – 3.14 cm (height), 1.12 cm (pseudostem diameter), 2.12 g (root mass) and 1.12 (total number of leaves plant⁻¹) – followed by isolate UPM31P1 (Fusarium oxysporum). The beneficial effect of UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum) was also reflected in the diseased plantlets, where pre-treatments with the isolates either singly (T6: UPM31P1; T8: UPM39B3) or in a mixture (T7: UPM31P1 + UPM39B3; T9: UPM14B1 + UPM13B8 + UPM39B3) were able to sustain the growth of plantlets, with significantly higher growth values than those in diseased plantlets that were not infected with endophytes (T10: FocR4). These results demonstrate the economic significance of these endophytic isolates, particularly UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum), both as potential growth promoters of banana and as agents rendering tolerance towards Fusarium wilt as a strategy in the management of Fusarium wilt of banana via improved vegetative growth.     

Silicon Suppresses Fusarium Wilt Development in Banana Plants

This study aimed to determine the effect of silicon (Si) in reducing the symptoms of Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), on banana plants. Banana seedlings of Grand Nain (resistant) and Maçã (susceptible) were grown in plastic trays amended with 0 (?Si) or 0.39 g Si (+Si) per kg of soil and inoculated with Foc at 60 days after transplanting. The Si concentration in the roots and rhizome‐pseudostem significantly increased by 30.26 and 58.82%, respectively, for the +Si treatment compared with ?Si treatment. The Si concentration in the roots and rhizome‐pseudostem of Grand Nain plants was, respectively, 11.57 and 37.04% greater than that found in Maçã. The +Si plants showed a reduction of 12.37, 49.81, 51.87 and 20.39%, respectively, for the area under reflex leaf symptoms progress curve, the area under root symptoms progress curve, the area under disease progress curve and the area under asymptomatic fungal colonization of tissue progress curve compared with ‐Si plants. The area under darkening of rhizome‐pseudostem progress curve (AUDRPPC) of Maçã significantly increased by 15.98% for the ?Si treatment in comparison with the +Si treatment. For the +Si treatment, the AUDRPPC of the plants from the Maçã cultivar significantly decreased by 20.59% in comparison with the plants from the Grand Nain cultivar. The area under relative lesion length progress curve (AURLLPC) of the plants from the Maçã cultivar significantly decreased by 41.54% for the +Si treatment in comparison with the ?Si treatment. There was no significant difference between the ‐Si and +Si treatments in the AUDRPPC and AURLLPC of Grand Nain. For the +Si treatment, the AURLLPC of Grand Nain significantly decreased by 9.23% in comparison with Maçã. There was no significant difference between the Grand Nain and Maçã for the AUDRPPC and AURLLPC in the ?Si treatment. The findings of this study show that supplying Si to banana plants, especially to a susceptible cultivar to Foc, had a great potential in reducing the intensity of Fusarium wilt and may play a key role in disease management when banana plants are cultivated in Si‐deficient soils infested by this pathogen.     

Response of endophytic bacterial communities in banana tissue culture plantlets to Fusarium wilt pathogen infection

Endophytic bacteria reside within plant hosts without having pathogenic effects, and various endophytes have been found to functionally benefit plant disease suppressive ability. In this study, the influence of banana plant stress on the endophytic bacterial communities, which was achieved by infection with the wilt pathogen Fusarium oxysporum f. sp. cubense, was examined by cultivation-independent denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA directly amplified from plant tissue DNA. Community analysis clearly demonstrated increased bacterial diversity in pathogen-infected plantlets compared to that in control plantlets. By sequencing, bands most similar to species of Bacillus and Pseudomonas showed high density in the pathogen-treated pattern. In vitro screening of the isolates for antagonistic activity against Fusarium wilt pathogen acquired three strains of endophytic bacteria which were found to match those species that obviously increased in the pathogen infection process; moreover, the most inhibitive strain could also interiorly colonize plantlets and perform antagonism. The evidence obtained from this work showed that antagonistic endophytic bacteria could be induced by the appearance of a host fungal pathogen and further be an ideal biological control agent to use in banana Fusarium wilt disease protection.     

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.     

香蕉植株根区土壤的真菌多样性分析

尖孢镰孢菌古巴专化型(Fusarium oxysporum f.sp.cubense)是香蕉枯萎病的病原菌,该菌是一种土壤习居菌,了解香蕉根区土壤中真菌多样性及镰孢菌属(Fusarium)真菌所占比例,对如何减少土壤中的病原菌、预防香蕉枯萎病的发生有重要的指导意义。该文通过采集不同宿根年限的香蕉健康植株和枯萎病植株的根区土壤,利用高通量测序技术测定土壤样品中的真菌种群。结果表明:(1)同一宿根年限的香蕉植株中,健康植株根区土壤中所获的reads及OTUs数量均高于枯萎病植株,说明健康植株根区土壤的真菌多样性丰富于枯萎病植株。(2)除了一年生香蕉枯萎病植株以担子菌门(Basidiomycota)为主外,其他土壤样品中均以子囊菌门(Ascomycota)为主,其中的丛赤壳科最高相对丰度来自三年生健康植株的根区土壤(26.02%),其次是五年生的枯萎病植株根区土壤(15.56%)。(3)在丛赤壳科中,镰孢菌属在三年生健康植株土壤中的相对丰度最高(2.54%),在其他样品中的相对丰度在0.1%～0.65%之间;在镰孢菌属中,腐皮镰孢菌(Fusarium solani)的相对丰度(0～1.59%之间)高于尖孢镰孢菌(F.oxysporum),尖孢镰孢菌仅占很小的比例(相对丰度0～0.08%之间)。可见,在不同香蕉植株的根区土壤中,健康植株的根区土壤真菌多样性高于枯萎病植株,无论是健康植株还是枯萎病植株的根区土壤中,作为香蕉枯萎病病原菌的镰孢菌属或尖孢镰孢菌的群体均不占主导地位。     

The application of high-throughput AFLP's in assessing genetic diversity in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is responsible for fusarium wilt of bananas. The pathogen consists of several variants that are divided into three races and 21 vegetative compatibility groups (VCGs). Several DNA-based techniques have previously been used to analyse the worldwide population of Foc, sometimes yielding results that were not always consistent. In this study, the high-resolution genotyping method of AFLP is introduced as a potentially effective molecular tool to investigate diversity in Foc at a genome-wide level. The population selected for this study included Foc isolates representing different VCGs and races, isolates of F. oxysporum f. sp. dianthi, a putatively non-pathogenic biological control strain F. oxysporum (Fo47), and F. circinatum. High-throughput AFLP analysis was attained using five different infrared dye-labelled primer combinations using a two-dye model 4200s LI-COR automated DNA analyser. An average of approx. 100 polymorphic loci were scored for each primer pair using the SAGA^MX automated AFLP analysis software. Data generated from five primer pair combinations were combined and subjected to distance analysis, which included the use of neighbour-joining and a bootstrap of 1000 replicates. A tree inferred from AFLP distance analysis revealed the polyphyletic nature of the Foc isolates, and seven genotypic groups could be identified. The results indicate that AFLP is a powerful tool to perform detailed analysis of genetic diversity in the banana pathogen Foc.     

施肥处理对不同抗性品种香蕉枯萎病的防控效果

【目的】随着香蕉枯萎病菌4号生理小种热带型(简称Foc TR4))在云南的入侵、传播和蔓延,对云南的香蕉产业产生严重的威胁。通过实时荧光定量PCR分析蕉园定植香蕉后7个月内的土壤中枯萎病病原菌TR4含量动态变化,明确不同香蕉品种的大田抗性表现以及不同肥料的防控效果,为枯萎病的防控提供技术参考。【方法】选用巴西蕉、桂蕉1号、南天黄和自主选育的云蕉1号为供试品种开展田间试验,设置虾肽有机肥+虾肽特护+虾肽果叶康(简称:虾肽有机肥处理)、常规有机肥+微生物制剂(简称:微生物处理)和常规有机肥(简称:对照)3个处理,调查4个品种在4个时间段的枯萎病发病率和3种肥料的防治效果。【结果】在月平均枯萎病病原菌TR4含量均超过2000拷贝的土壤条件下,4个品种的发病率在3个施肥处理中均表现出差异性,南天黄、云蕉1号的发病率与其他2个主栽感病品种的发病率差异达显著水平;3种施肥处理间的发病率达显著差异,发病率从高到低表现为对照虾肽有机肥处理微生物处理。【结论】施用微生物制剂对降低枯萎病发病率起一定的作用。南天黄的抗病性较强,云蕉1号也表现出较强的抗性,但还有待进一步改良和提高抗性。     

Plant-growth-promoting traits and antifungal potential of the Bacillus amyloliquefaciens YL-25

Biological control of plant soil-borne diseases has been shown as an attractive and an environment friendly alternative to chemical fungicides. Different microbial strains have been reported effective in controlling plant pathogens. Among those, Bacillus strains have their own importance. Bacillus amyloliquefaciens strain YL-25, isolated from the rhizosphere of healthy banana plant, was evaluated as bio-organic fertiliser (BIO) for its ability to promote plant growth and suppress Fusarium wilt of banana in pot experiment. The results showed that the application of the BIO containing strain YL-25 significantly promoted the growth of banana plants and decreased the incidence of Fusarium wilt compared to the organic fertiliser and chemical fertiliser (CF). In order to explore the beneficial mechanisms of strain YL-25, experiments were conducted in vitro. The phytohormones including indole-3-acetic acid and gibberellin A3 and stable antifungal compounds three homologous of iturin A were identified in the culture broth of strain YL-25. The strain YL-25 also showed the ability to degrade extracellular phytate in plate experiment. Owing to its innate multiple functional traits and biocontrol activity, the strain YL-25 may be used as plant-growth-promoting rhizobacterium and biocontrol agent against Fusarium wilt of banana.     

Systemic acquired resistance in Cavendish banana induced by infection with an incompatible strain of Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fact that there are no economically viable biological, chemical, or cultural measures of controlling the disease in an infected field leads to search for alternative strategies involving activation of the plant's innate defense system. The mechanisms underlying systemic acquired resistance (SAR) are much less understood in monocots than in dicots. Since systemic protection of plants by attenuated or avirulent pathogens is a typical SAR response, the establishment of a biologically induced SAR model in banana is helpful to investigate the mechanism of SAR to Fusarium wilt. This paper described one such model using incompatible Foc race 1 to induce resistance against Foc tropical race 4 in an in vitro pathosystem. Consistent with the observation that the SAR provided the highest level of protection when the time interval between primary infection and challenge inoculation was 10 d, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL, EC 4.3.1.5), peroxidase (POD, EC 1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), and superoxide dismutase (SOD, EC 1.15.1.1) in systemic tissues also reached the maximum level and were 2.00–2.43 times higher than that of the corresponding controls on the tenth day. The total salicylic acid (SA) content in roots of banana plantlets increased from about 1 to more than 5 μg g⁻¹ FW after the second leaf being inoculated with Foc race 1. The systemic up-regulation of MaNPR1A and MaNPR1B was followed by the second up-regulation of PR-1 and PR-3. Although SA and jasmonic acid (JA)/ethylene (ET) signaling are mostly antagonistic, systemic expression of PR genes regulated by different signaling pathways were simultaneously up-regulated after primary infection, indicating that both pathways are involved in the activation of the SAR.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

Acremonium as an endophytic bioagent against date palm Fusarium wilt

A total of 250 endophytic fungal isolates, representing 30 morphotaxa, were isolated and characterised, they were collected from the different living symptomless parts of date palm trees of orchards of six Egyptian governorates. Colonisation was greater in samples from the midrib than in those from laminar tissue and slightly greater at the tip of the lamina compared with the base of the leaf. Acremonium spp. were frequently isolated as date palm root endophytes. Acremonium isolates were screened in Petri dishes to select the highest antagonistic one against an Algerian isolate of Fusarium oxysporum f.sp. albedinis. Two-week-old axenically reared date palm seedlings grown in Petri dishes were directly injected with spore suspension (1.5?×?10⁷ spores/ml) of a pure culture of the virulent antagonistic isolate of Acremonium sp. One week after endophytic colonisation, date palm seedlings were then challenged with the pathogen, Fusarium albedinis. The challenged seedlings exhibited a significant reduction in wilt symptom percentage (by 87.0%), while the seedlings exposed to Fusarial toxin without pathogen exhibited the wilt disease symptoms. This indicates that the endophyte ably depresses any toxic action of F. albedinis. The endophytic fungus was recovered from sites distant from the point of inoculation after six?months from the application, indicating that the Acremonium sp. has the potential to move throughout the tissue plant, even the end time of trial. The Acremonium mode of action, as a biocontrol agent, was discussed.     

Dual inoculation of Fusarium oxysporum endophytes in banana: effect on plant colonization,growth and control of the root burrowing nematode and the banana weevil

The burrowing nematode (Radopholus similis (Cobb) Thorne) and the banana weevil (Cosmopolites sordidus Germar, Coleoptera: Curculionidae) are major pests of banana (Musa spp.) in the Lake Victoria basin region of Uganda. Among biological options to control the two pests is the use of non-pathogenic Fusarium oxysporum Schltdl.: Fries endophytes of banana. We investigated the ability of endophytic F. oxysporum isolates Emb2.4o and V5w2 to control the banana weevil and the burrowing nematode, alone and in combination. Plant colonization by the endophytes was determined by inoculating their chemical-resistant mutants separately and in combination, onto banana roots. Plant growth promotion was determined by measuring plant height, girth, number of live roots and fresh root weight at harvest, and control of the nematode and weevil was determined by challenging endophyte-inoculated plants with the pests 8 weeks after endophyte inoculation. Endophytic root colonization was highest in plants inoculated with both endophytes, compared with those inoculated with only one of the endophytes. Root colonization was better for isolate V5w2 than Emb2.4o. Dually inoculated plants showed a significant increase in height, girth, fresh root weight and number of functional roots following nematode challenge. Nematode numbers in roots were reduced 12 weeks after challenge of 8-week-old endophyte-inoculated plants. Significant reductions in weevil damage were observed in the rhizome periphery, inner and outer rhizomes, compared with endophyte non-inoculated controls. We conclude that dual inoculation of bananas with endophytic isolates Emb2.4o and V5w2 increases root colonization by the endophytes, reduces nematode numbers and weevil damage, and enhances plant growth in the presence of nematode infestation.     

Biological Control Activity of Three Trichoderma Isolates Against Fusarium Wilts of Cotton and Muskmelon and Lack of Correlation with their Lytic Enzymes

The enzymatic activity and the biocontrol ability of two new isolates of Trichoderma spp. (T-68 and Gh-2) were compared in laboratory and glasshouse experiments with a previously studied T. harzianum strain (T-35). In dual culture tests with Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. vasinfectum, isolates T-68 and Gh-2 overgrew the colonies of Fusarium, whereas T-35 failed to parasitize both wilt pathogens. Under glasshouse conditions, the three isolates of Trichoderma were effective in controlling Fusarium wilt of cotton but only T-35 was effective against F. oxysporum f. sp. melonis on muskmelon. When the three Trichoderma isolates were grown on liquid media containing laminarin, colloidal chitin or F. oxysporum f. sp. melonis cell walls as sole carbon sources, maximum β-1,3-glucanase and chitinase specific activity in the culture filtrates of all fungi was reached after 72h of incubation. When culture filtrates of the three Trichoderma isolates were incubated with freeze-dried mycelium of F. oxysporum f. sp. melonis or F. oxysporum f. sp. vasinfectum, different concentrations of glucose and N-acetyl-D-glucosamine were released. Overall no correlation was found between enzymatic activity and the biocontrol capability against Fusarium wilt on muskmelon and cotton.     

Modified Primers for the Identification of Nonpathogenic Fusarium oxysporum Isolates That Have Biological Control Potential against Fusarium Wilt of Cucumber in Taiwan

Previous investigations demonstrated that Fusarium oxysporum (Fo), which is not pathogenic to cucumbers, could serve as a biological control agent for managing Fusarium wilt of cucumber caused by Fo f. sp. cucumerinum (Foc) in Taiwan. However, thus far it has not been possible to separate the populations of pathogenic Fo from the nonpathogenic isolates that have biological control potential through their morphological characteristics. Although these two populations can be distinguished from one another using a bioassay, the work is laborious and time-consuming. In this study, a fragment of the intergenic spacer (IGS) region of ribosomal DNA from an Fo biological control agent, Fo366, was PCR-amplified with published general primers, FIGS11/FIGS12 and sequenced. A new primer, NPIGS-R, which was designed based on the IGS sequence, was paired with the FIGS11 primer. These primers were then evaluated for their specificity to amplify DNA from nonpathogenic Fo isolates that have biological control potential. The results showed that the modified primer pair, FIGS11/NPIGS-R, amplified a 500-bp DNA fragment from five of seven nonpathogenic Fo isolates. These five Fo isolates delayed symptom development of cucumber Fusarium wilt in greenhouse bioassay tests. Seventy-seven Fo isolates were obtained from the soil and plant tissues and then subjected to amplification using the modified primer pair; six samples showed positive amplification. These six isolates did not cause symptoms on cucumber seedlings when grown in peat moss infested with the isolates and delayed disease development when the same plants were subsequently inoculated with a virulent isolate of Foc. Therefore, the modified primer pair may prove useful for the identification of Fo isolates that are nonpathogenic to cucumber which can potentially act as biocontrol agents for Fusarium wilt of cucumber.     

Organic matter fractions by SP-MAS C NMR and microbial communities involved in the suppression of Fusarium wilt in organic growth media

Fusarium wilt is an economically important disease in carnation and tomato plants. The use of suppressive plant growth media has become an alternative method for plant disease control due to the lack of effective chemical control measures. Plant disease suppressiveness is sustained only in plant growth media with an adequate organic matter (OM) composition. Carbohydrate polymers are the most important sources of carbon nutrient for microbial community in these media, mainly consisting of cellulose and hemicellulose. This determines microbial activity, biomass and selects microbial communities in plant growth media, which are reported factors associated with Fusarium wilt suppressiveness.This work determined OM carbon functional groups using Single Pulse Magic Angle Spinning ¹³C-Nuclear Magnetic Resonance (SP-MAS ¹³C-NMR) in three plant growth media with different suppressiveness levels to Fusarium wilt in two crops, carnation and tomato. We propose that the critical role of OM to sustain naturally occurring suppressiveness in those media is not related with cellulose reserve. This could be explained because cellulose protected by lignin encrustation is not available to microbial degradation, meaning that cellulose availability is critical to sustenance of microorganism-mediated biological control. However, the hemicellulose relative abundance (peak 175 ppm) was associated to Fusarium wilt suppression level in plant growth media studied.Carbon source availability in OM was related to microbial biomass and econutritional group population densities involved in biocontrol. For these composts, Bacillus spp., oligotrophic and cellulolytic actinomycetes, and oligotrophic actinomycetes/oligotrophic bacteria and cellulolytic actinomycetes/cellulolytic bacteria ratios were indicated as microbial populations potentially involved in suppression.