Studies on antagonism betweenFusarium udum Butler and root region microflora of pigeon-pea

Antagonism betweenFusarium udum Butler causing wilt of pigeon-pea (Cajanus cajan (L.) Millsp.) and the saprophytic microflora of the root region of the host was studied with reference to colony interaction, hyphal interference, volatile and non-volatile metabolites and staling growth products. Studies were extended to screen potential antagonists against the wilt pathogen in soil. Aspergillus flavus, A. niger, A. terreus, Penicillium citrinum andMicromonospora globosa (an actinomycete) were antagonistic againstF. udum, whereas the pathogen parasitized and killedAspergillus luchuensis, Cunninghamella echinulata, Curvularia lunata, Mortierella subtilissima andSyncephalastrum racemosum. The pattern of growth of microorganisms on nutrient agar staled by rhizosphere soil inocula of healthy or wilted pigeon-pea plants was found to be different.F. udum colonized and grew on nutrient agar staled by the rhizosphere inoculum of the wilted plants upto 120h of incubation. However, it could not colonise and grow on the nutrient agar staled by rhizosphere microflora of healthy plants after 48h of incubation because of the presence of antagonists likeA. niger, A. flavus, A. terreus and a few species ofPenicillium in the soil inoculum. When pure cultures in soil ofF. udum was mixed with those of antagonists in different ratios,A. niger, A. flavus andM. globosa significantly suppressed the population ofF. udum, whereasA. terreus markedly reduced the population. When inoculated in soil, the antagonists exhibited a high fungistatic activity againstF. udum.     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

Effect of inoculum density,nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Summary Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH₄–N than in media fertilized with NO₃–N.Portion of M. S. thesis submitted by the senior author to the Graduate School, University of Florida, Gainesville. Florida Agricultural Experiment Stations Journal Series No. 4221.     

Formation of resting body byCephalosporium roseo-griseum Saksena inside the conidiophore ofAspergillus flavus Link. Ex Fries during mycoparasitism

Summary Mycoparasitic behaviour ofCephalosporium roseo-griseum Saksena withAspergillus flavus Link. ex Fries was studiedin vitro. Frequent coiling of hyphae ofC. roseo-griseum around the conidiophores ofA. flavus, penetration of hyphae ofC. roseo-griseum inside those ofA. flavus, and formation of resting bodies byC. roseo-griseum in the conidiophores ofA. flavus were observed.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Interaction between pathogenic and saprobic fungi isolated from soybean roots and seeds

A variety of interactions was recorded in culture between 11 saprobic fungi isolated from soybean (Glycine max) roots and seeds and the soybean pathogens Cercospora sojina, Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae, and Septoria glycines. The most active saprobes were Aspergillus terreus, Chaetomium cupreum, Epicoccum nigrum, Gliocladium roseum, Myrothecium roridum, Penicillium thomii, and Trichothecium roseum. Hyphal lysis of several fungal pathogens by Acremonium sp., C. cupreum and P. thomii was recorded perhaps because of parasitism by G. roseum and T. roseum. In greenhouse studies, seeds coated with G. roseum, P. thomii, and T. harzianum emerged significantly (P=0.05) more than those coated with A. terreus and the control. In field studies, seeds coated with a conidial suspension of A. terreus, G. roseum, P. thomii or Trichoderma harzianum produced a significantly greater stand than the control. The area of cotyledons covered with lesions caused by C. truncatum was significantly less on seeds coated with G. roseum, P. thomii and T. harzianum than the control.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

Conidial surface ultrastructure of human-pathogenic and saprobicCladosporium species

Freeze-fracturing of outer wall layers ofCladosporium conidia revealed two types of ultrastructure, coinciding with taxonomic characteristics. The outer conidial layers were essentially smooth in the human pathogenic species,C. bantianum, C. carrionii, andC. trichoides. In contrast, mosaic arrays of rodlets on conidia were observed with freeze-fracturing in the saprobic species,C. cladosporioides, C. coralloides, C. herbarum, C. sphaerospermum, andC. variabile. Conidia ofC. elatum were an exception among the saprobic species as they had smooth surfaces. The present study supports the suggestion that the human pathogenicCladosporium species should be transferred to another genus.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Effects of Commercial and Indigenous Microorganisms on Fusarium Wilt Development in Chickpea

The purpose of this research was to determine whetherBacillus subtilis,nonpathogenicFusarium oxysporum,and/orTrichoderma harzianum,applied alone or in combination to chickpea (Cicer arietinumL.) cultivars ‘ICCV 4’ and ‘PV 61’ differing in their levels of resistance to Fusarium wilt, could effectively suppress disease caused by the highly virulent race 5 ofFusarium oxysporumf. sp.ciceris.Seeds of both cultivars were sown in soil amended with the three microbial antagonists, alone or in combination, and 7 days later seedlings were transplanted into soil infested with the pathogen. All three antagonistic microorganisms effectively colonized the roots of both chickpea cultivars, whether alone or in combination, and significantly suppressed Fusarium wilt development. In comparison with the control, the incubation period for the disease was delayed on average about 3 days and the final disease severity index and standardized area under the disease progress curve were reduced significantly between 14 and 33% and 16 and 42%, respectively, by all three microbial antagonists. Final disease incidence only was reduced byB. subtilis(18–25%) or nonpathogenicF. oxysporum(18%). The extent of disease suppression was higher and more consistent in ‘PV 61’ than in ‘ICCV 4’ whether colonized byB. subtilis,nonpathogenicF. oxysporum,orT. harzianum.The combination ofB. subtilis+T. harzianumwas effective in suppressing Fusarium wilt development but it did not differ significantly from treatments with either of these antagonists alone. In contrast, the combination ofB. subtilis+ nonpathogenicF. oxysporumtreatment was not effective but either antagonist alone significantly reduced disease development.     

Responses of four arid zone grass species from varying habitats to drought stress

The effects of 4 or 8 drought cycles on four grass species,Cenchrus pennisetiformis, Leptochloa fusca, Panicum turgidum, andPennisetum divisum were assessed in a pot experiment. There were significant differences between the species in biomass production under water stress.C. pennisetiformis andP. turgidum produced significantly greater fresh and dry matter thanP. divisum and especially thanL. fusca. L. fusca had the lowest andP. divisum highest osmotic potentials compared with the other species after the completion of 4 or 8 drought cycles. Osmotic adjustment (difference between osmotic potential of droughted/rehydrated plants and control plants) was highest inL. fusca. The stomatal conductance was significantly decreased with increased drought stress inC. pennisetiformis. The elasticity ofC. pennisetiformis, P. turgidum andP. divisum increased with increase in number of drought cycles, whereas that ofL. fusca remained unchanged.L. fusca andP. turgidum had the lowest leaf hydration of all species after 8 drought cycles. The chlorophyllsa andb in all species remained unaffected by drought treatments. The proline content ofC. pennisetiformis andL. fusca increased significantly with increased drought stress, whereas that ofP. turgidum remained unaffected after 4 or 8 drought cycles.L. fusca synthesized great amount of leaf soluble proteins during 8 drought cycles, whereasP. divisum had low protein content after 4 drought cycles. The protein contents ofC. pennisetiformis andP. turgidum remained unaffected after 8 drought cycles. The leaf epicuticular wax ofL. fusca increased consistently with increased drought stress, but leaf wax ofP. divisum increased only at the highest drought stress and that ofC. pennisetiformis andP. turgidum increased after 4 drought cycles. On the basis of these results it was established thatC. pennisetiformis andP. turgidum were the most tolerant,P. divisum intermediate, andL. fusca the most sensitive to drought stress. The osmotic adjustment did not positively correlate with the degree of drought resistance.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Interrelationship betweenCoprinus lagopus andFusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism

Summary The interrelationship between the growth ofCoprinus lagopus andFusarium udum on pigeon pea substrates in soil was studied at 22-2°C and 30±2°C in relation to their competitive saprophytic ability and antagonism.C. lagopus was observed to be a potent and frequent colonizer of pigeon pea substrates in soil along withF. udum. Saprophytic colonization of pigeon pea substrate byF. udum precolonized byC. lagopus was inhibited in all inoculum soil mixtures. Saprophytic colonization of the substrate byF. udum was better at 22°C than at 30°C and that byC. lagopus better at 30°C than at 22°C. The colonization of substrate by each fungus was suppressed by the other fungus depending upon the temperature used. Hyphal parasitism and colony interactions between these fungi and also with a number of other saprophytic fungi were investigated. No antagonistic reaction was observed betweenF. udum andC. lagopus. However, these fungi were observed to be potent antagonists against other microfungi tested. The formation of fruiting bodies ofC. lagopus was also observed in the laboratory on nutrient media but more frequently on pigeon pea substrates.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.     

Phylogenetic relationships ofCryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences

The genusCryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic speciesC. neoformans, comprising 4 serotypes and havingFilobasidiella neoformans andF. bacillispora as teleomorphs, was found at a relatively large distance fromFilobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A.Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered withF. neoformans.The genusFilobasidium was clearly separated fromFilobasidiella and clustered withC. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus andC. ater. The latter may represent the anamorph ofFilobasidium elegans. The organe to red species ofCryptococcus, as well asC. aquaticus andC. yarrowii, were found completely unrelated with these taxa,C. macerans being affiliated toCystofilobasidium capitatum.The genusTrichosporon was found relatively homogeneous; it includesC. humicola, C. curvatus and the filamentous speciesHyalodendron lignicola. Cryptococcus flavus andC. dimennae probably belong to the Tremellales, though distances between these species are large. The positions ofC. laurentii andC. luteolus remains to be determined.     

Peanut-pod mycoflora and kernel infection

Summary The mycoflora in soil clinging to dry pods of peanuts of the Spanish variety Argentine was sampled in 2 experiments by serially washing pods for increasing periods in changes of sterile water. Of the 9 principal fungi found,Aspergillus niger, A. flavus, A. terreus, Rhizopus spp. andSclerotium bataticola were present initially in relatively small numbers and decreased rapidly in subsequent dilutions. This decrease paralleled a decrease in weight of suspended material and in percentage of soil and organic particles greater than 0.016 mm in size.Penicillium funiculosum, P. rubrum, P. citrinum, andFusarium spp. were found in large numbers and increased or slowly decreased in numbers in subsequent dilutions. In some instances variations in numbers followed trends of percentages of soil and organic particles less than 0.016 mm in size.When dry pods with this known mycoflora were allowed to hydrate over a 6-day period at 26°, 32°, or 38°C, there was extensive pod penetration and kernel infection byA. niger, A. flavus, S. bataticola andRhizopus spp. but not by other fungi. The degree ofA. flavus andA. niger infection increased with increasing temperatures.Approved by the Director as Journal Series Paper No. 135.     

Inter-relationship between shoot weight,severity of root rot and survival rate of subterranean clover inoculated with certain pathogenic fungi

Summary Plant survival rate, root disease index and fresh shoot weight of subterranean clover seedlings inoculated with fungal pathogens (Fusarium avenaceum, F. oxysporum, Phoma medicaginis, Pythium irregulare andRhizoctonia solani, both singly and in combinations) were generally inter-correlated over a wide range of temperature (10, 15, 20 and 25°C) and moisture conditions (45, 65% water holding capacity and flooding). There was a negative correlation between root disease index and shoot weight for all treatments exceptF. avenaceum + P. irregulare. Root disease index and seedling survival rate were negatively correlated except forF. oxygsporum, Phoma medicaginis, P. irregulare andF. oxysporum + F. avenaceum. However, a good positive correlation was found between the survival rate and shoot weight for all treatments with the exception ofPhoma medicaginis.     

Microsatellite marker-based detection of Fusarium wilt pathogens of Psidium guajava L.

Wilt of Psidium guajava L., incited by Fusarium oxysporum f. sp. psidii and Fusarium solani is a serious soil-borne disease of guava in India. Forty-two isolates each of F. oxysporum f. sp. psidii (Fop) and F. solani (Fs) collected from different agro climatic zones of India showing pathogenicity were subjected to estimate the genetic and molecular characterisation in terms of analysis of microsatellite marker studies. Out of eight microsatellite markers, only four microsatellite markers, viz. MB 13, MB 17, RE 102 and AY212027 were amplified with single band pattern showing the character of identical marker for molecular characterisation and genetic identification. Microsatellite marker MB 13 was amplified in F. oxysporum f. sp. psidii and F. solani isolates. Product size of 296 bps and 1018 bps were exactly amplified with a single banding pattern in all the isolates of F. oxysporum f. sp. psidii and F. solani, respectively. Microsatellite markers, viz. MB 17, RE 102 and AY212027 were also exactly amplified with a single banding pattern. MB 17 was amplified in F. oxysporum f. sp. psidii isolates with a product size of 300 bp. RE 102 and AY212027 were amplified in F. solani isolates with the product size of 153 bp and 300 bp, respectively. Therefore, amplified microsatellite marker may be used as identifying DNA marker.     

Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus

The biological control efficacy of single or multiple applications of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus toward Radopholus similis was investigated in pot trials with banana under glasshouse conditions. R. similis was controlled substantially in single and combined applications of F. oxysporum with P. lilacinus or B. firmus. The combination of F. oxysporum and P. lilacinus caused a 68.5% reduction in nematode density whereas the individual applications reduced the density by 27.8% and 54.8% over the controls, respectively. Combined application of F. oxysporum and B. firmus was the most effective treatment in controlling R. similis on banana (86.2%), followed by B. firmus alone (63.7%). The compatibility of the biocontrol agents, as well the capacity of F. oxysporum to colonize banana roots in the absence or presence of P. lilacinus was also investigated. P. lilacinus did not adversely affect endophytic colonization by F. oxysporum. Biological control of R. similis in banana can therefore be enhanced via combined applications of antagonists with different modes of action that target different stages in the infection process. Handling editor: Ralf-Udo Ehlers