Isolation, Identification, and Activity of Mycoherbicidal Pathogens from Juvenile Broomrape Plants

Although there are reports of isolation of mycoherbicidal pathogens attacking the widespread broomrapes (Orobanche spp.) that parasitize legumes and vegetables, none is in use or available. This is despite there being no good method of controlling broomrapes in most crops other than by preplant fumigation with methyl bromide. Two highly parasitic fungi, Fusarium arthrosporioides strain E4a (CNCM I-164) and F. oxysporum strain E1d (CNCM I-1622), were isolated from nearly 100 organisms found on diseased, juvenile, emerging Orobanche flower stalks. A near-axenic polyethylene envelope system for culturing broomrape on tomato roots was used to ascertain pathogenicity of these strains. Both organisms fulfilled Koch's postulates for being primary pathogens. Their DNAs were analyzed and fingerprinted by restriction fragment length polymorphism and random amplified polymorphic DNA, showing that they are indeed different from each other and from many other Fusarium spp. and other formae speciales of F. oxysporum including a strain that attacks O. cumana on sunflowers. Both strains infect O. aegyptiaca, O. cernua, and O. ramosa, but not O. cumana. They did not infect any of the vegetable and legume crops tested and thus seem specific to Orobanche. Tomato plant roots dipped into a fungal spore and mycelial suspension and planted in broomrape-infested soil were protected for 6 weeks, as were tomato transplants in pot experiments. About 90% control was also achieved by posttransplant soil drench with fungal suspensions in pot experiments. These pathogens may be effective as seed, transplant, or soil-drench treatments of high-value vegetable and other crops.     

Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins

A total of 57 samples of feedstuffs commonly used for animal nutrition in Colombia (corn, soybean, sorghum, cottonseed meal, sunflower seed meal, wheat middlings and rice) were analyzed for Fusarium contamination. Fusarium fungi were identified at species level by means of conventional methods and the ability to produce fumonisins of the most prevailing species was determined. A total of 41 of the feedstuffs analyzed (71.9%) were found to contain Fusarium spp. Most contaminated substrates were corn (100%), cottonseed meal (100%), sorghum (80%), and soybean (80%). Wheat middlings and rice showed lower levels of contamination (40% and 20%, respectively), while no Fusarium spp. could be isolated from sunflower seed meal. The most prevalent species of Fusarium isolated were F. verticilliodes (70.8%), F.␣proliferatum (25.0%), and F. subglutinans (4.2%). All of them correspond to section Liseola.Production of fumonisins on corn by the isolated Fusarium was screened through liquid chromatography. Almost all strains of F. verticilliodes (97.1%) produced FB1 (5.6–25,846.4 mg/kg) and FB2 (3.4–7507.5 mg/kg). Similarly, almost all strains of F.␣proliferatum (91.7%) produced fumonisins but at lower levels than F.␣verticilliodes (FB1 from 6.9 to 3885.0 mg/kg, and FB2 from 34.3 to 373.8 mg/kg), while F. subglutinans did not produce these toxins. This is the first study in Colombia describing toxigenic Fusarium isolates from␣animal feedstuffs.     

Survey ofFusarium species in an arid environment of Bahrain

The prevalence and distribution of soil-inhabitingFusarium species in the soil of vegetable crops grown under custom-made plastic tunnels were studied at three geographical locations on the island of Bahrain. Six species ofFusarium representing a total of 1154 isolates were isolated on modified Komada medium. All species reported in this survey are recorded for the first time from the hot, arid desert of Bahrain.Fusarium solani andF. oxysporum were among the most frequently isolated fungi in all locations and crops.     

Land use intensification affects soil microbial populations, functional diversity and related suppressiveness of cucumber Fusarium wilt in China’s Yangtze River Delta

The extent of soil microbial diversity in agricultural soils is critical to the maintenance of soil health and quality. The aim of this study was to investigate the influence of land use intensification on soil microbial diversity and thus the level of soil suppressiveness of cucumber Fusarium wilt. We examined three typical microbial populations, Bacillus spp., Pseudomonas spp. and Fuasarium oxysporum, and bacterial functional diversity in soils from three different land use types in China’s Yangtze River Delta, and related those to suppressiveness of cucumber Fusarium wilt. The land use types were a traditional rice wheat (or rape) rotation land, an open field vegetable land, and a polytunnel greenhouse vegetable land that had been transformed from the above two land use types since 1995. Results generated from the field soils showed similar counts for Bacillus spp. (log 5.87–6.01 CFU g⁻¹ dw soil) among the three soils of different land use types, significantly lower counts for Pseudomonas spp. (log 5.44 CFU g⁻¹ dw soil) in the polytunnel greenhouse vegetable land whilst significantly lower counts for Fusarium oxysporum (log 3.21 CFU g⁻¹ dw soil) in the traditional rice wheat (or rape) rotation land. A significant lower dehydrogenase activity (33.56 mg TPF kg⁻¹ dw day⁻¹) was observed in the polytunnel greenhouse vegetable land. Community level physiological profiles (CLPP) of the bacterial communities in soils showed that the average well color development (AWCD) and three functional diversity indices of Shannon index (H′), Simpson index (D) and McIntosh index (U) at 96 h incubation in BIOLOG Eco Micro plates were significantly lower in the polytunnel greenhouse vegetable land than in both the traditional rice wheat (or rape) rotation land and the open field vegetable land. A further greenhouse experiment with the air-dried and sieved soils displayed significantly lower plant growth parameters of 10-old cucumber seedlings as well as significantly lower biomass and total fresh fruit yield at the end of harvesting at day 70 in the polytunnel greenhouse vegetable soil sources. The percentages of Fusarium wilt plant death were greatly increased in the polytunnel greenhouse vegetable plants, irrespective of being inoculated with or without Fusarium oxysporum f. sp. cucumerinum. Our results could provide a better understanding of the effects of land use intensification on soil microbial population and functional diversity as well as the level of soil suppressiveness of cucumber Fusarium wilt.     

Seasonal variations of Fusarium species in wheat fields in Upper Egypt

Abstract

Populations of the genus Fusarium in wheat fields were studied within the crop-growing season at Qena area (Upper Egypt) using two different types of media (DCPA and DRBA) at 25°C. Fourteen Fusarium species were isolated during this study, namely F. anthophilum, F. aquaeductuum, F. chlamdosporum, F. dimerum, F. merismoides, F. moniliforme, F. oxysporum, F. poae, F. proliferatum, F. sambucinum, F. scripi, F. solani, F. sporotrichioides and F. subglutinans. Fusarium merismoides, F. oxysporum and F. sambucinum were the most common Fusarium species isolated from different wheat plant parts (rhizosphere and rhizoplane) as well as from the wheat fields (soil and air). Fusarium spp. rarely appeared at the beginning of the season and increased sharply between January to March and decreased slightly or sharply at the end of the season according to the type of media and isolation source.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20–35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.     

Occurrence and distribution of Fusarium species in maize fields in New Zealand

Fusarium populations were investigated in maize grains and their husks about six weeks before harvest in three maize fields in the Manawatu region of New Zealand. The role of litter and soil as reservoirs for these fungi was also examined. Two techniques were used to examine populations, dilution plating and direct plating. Using the dilution plating technique the highest overall populations were found in husks (mean 2.2 × 10⁵/g) and litter (mean 1.4 × 10⁵/g), while similar lower numbers of viable propagules were obtained from grain (mean 2.1 × 10³/g) and soil (2.8 × 10³/g). With this technique five Fusarium spp. were commonly isolated; F. graminearum (Gibberella zeae), F. culmorum, F. subglutinans, F. oxysporum and F. acuminatum, of which F. graminearum was the most abundant. With the direct plating technique 87% of grains were infected with Fusarium spp., with some grains being infected with more than one species. Segments from husks and litter, 70% and 43% respectively, were colonised by Fusarium spp. F. graminearum was the most frequent species isolated from maize grain and husk segments(48.3 and 37.7% colonisation respectively). Other species, particularly F. culmorum and F. acuminatum, were also found to be common contaminants. A total of 15 Fusarium spp. was recovered from all material examined by both techniques. Cultures with characteristics resembling those of F. moniliforme were rarely observed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Studies on the transmission of some Fusarium spp. to potato progeny tubers from mother plants and its biological control

Fusarium spp. attack potato roots causing root-rot, damping-off and wilt disease in Assuit Governorate. Forty-five Fusarium isolates were isolated from F. nygamai, F. acutatum, F. solani, F. proliferatum, F. subglutinans, and F. oxysporum. Isolates were tested for their pathogenic capability on Burn potato variety during growing season 2007/2008. Isolates infect potato plants causing either damping-off or wilt symptoms. Isolates varied in their virulence. Role of potato tuber seed in the transmission of the causal pathogen to daughter using Electrophoresis. Protein profiles of the tested isolates divided into four sub-clusters at similarity levels 93.79, 91.55 and 92.62% while isolate of Fusarium profile No. 11 formed separate sub-clusters at similarity level 69.79%. F. nygamai and F. solani were notable exception because profile No. 4 of F. nygamai from roots and profile No. 4 from sprouts were almost identical (similarity level 96.81%); similarity level between profile No. 8 from roots and profile no/8 from sprouts was 95.44%. Results prove that F. nygamai and F. solani are potato tuber seed-borne fungus. T. harzianum, T. viride, T. longibrachiatum, G. virens and E. nigrum or its filtrate inhibited the growth of F. nygamai, F. acutatum, F. solani, F. proliferatum, F. subglutinans and F. oxysporum. The formulation of T. harzianum, T. longibrachiatum and G. virens against tested pathogenic fungi reduce disease incidence under greenhouse conditions.     

Occurrence and distribution of <Emphasis Type="Italic">Microdochium nivale</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from barley,durum and soft wheat grains in France from 2000 to 2002

Fusarium Head Blight of small grain cereal is a disease of growing concern in Europe. Along with Microdochium nivale, several species of Fusarium may be associated with the disease, including species that are potentially toxigenic. This paper describes the results of a large scale survey of the variety and frequency of different Fusarium species and M. nivale in France. A total of 749 soft wheat, durum wheat and barley samples were collected and analyzed from 2000 to 2002. The most frequent species isolated were F.graminearum, F. avenaceum and F. poae. The frequency of F. poae seems to have increased while M.nivale and F. culmorum appear less frequent than previously described in France. Other Fusarium species detected in decreasing prevalence were F. tricinctum, F. equiseti, F. acuminatum, F. sambucinum, F.sporotrichioides, F. moniliforme, F. heterosporum, F. subglutinans and F. oxysporum. All the most frequent pathogenic species and also the less pathogenic ones were frequently associated with individual fields. The implications of these associations for the protection of cereals crops and for contamination by mycotoxins are discussed.     

Aggressiveness spectrum and genetic variability of Fusarium spp. on rice and wheat varieties

Abstract

A total of 106 Fusarium spp. were isolated from infected roots and soil samples of wheat and rice. Of the 106 isolates, 32 from wheat, and 74 from rice, were isolated. Six Fusarium spp. (F. oxysporum, F. moniliforme, F. poae, F. graminearum, F. tricinctum and F. equiseti) were identified at specie level. In aggressiveness tests Fusarium spp. root rot causing fungi were screened out into different aggressiveness classes according to disease severity scales. The aggressiveness of Fusarium spp. was studied on wheat varieties (Inqalab-91 and chakwal-86) and on rice varieties (Basmati-385 and IRRI-6) under controlled conditions. The overall total number of aggressive isolates was higher in rice than in wheat. However, the percentage of severely aggressive isolates was high in wheat, whereas the percentage of moderately and slightly aggressiveness isolates was high in rice. In rice, five isolates were non-aggressive and on wheat 17 were non-aggressive. Random Amplified Polymorphism DNAs (RAPDs) were used to study the polymorphism and genetic variations within the population of Fusarium spp. that established to study correlation between taxonomical and genetical characters of fungi. Five random primers were used P1 (5′-AGGAGGACCC-3′), P2 (5′-ACGAGGGACT-3′), PE7 (5′-AGATGCAGCC-3′), P14 (5′-CCACAGCACG-3′) and PE20 (5′-AACGGTGACC-3′). Each of the 10-mer primers produced results based on the respective banding patterns they generated in present investigations. Primers distinguished the F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti. All the tested primers yielded amplification products, and that were reproducible. Although there was some intraspecific variation with primers, some strains were similar and some were different in banding pattern. In F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti were seen clustered close to one another but each primer separated them unambiguously. All primer (P1, P2, P14, PE7 and PE20) combination produced 62 bands. All primers have shown interspecific and intraspecific variations in banding patterns.     

The distribution ofFusarium species in soils planted to millet and sorghum in Lesotho,Nigeria and Zimbabwe

Samples from soils planted to millet and sorghum from Lesotho, Nigeria, and Zimbabwe were processed and a total of 3,291Fusarium cultures were recovered. Of these 1,296 cultures were isolated from plant debris and 1,995 cultures were recovered from soil dilutions. The most prevalent species recovered wereF. oxysporum (37%),F. equiset (30%),F. solani (14%),F. moniliforme (6%),F. compactum (5%),F. nygamai (4%), andF. chlamydosporum (2%). OtherFusarium species isolated wereF. merismoides, F. polyphialidicum, F. graminearum, F. subglutinans, F. sambucinum, F. longipes, F. semitectum, F. dimerum, F. lateritium, and a group of cultures designated as population A which resembleF. camptoceras. Fusarium equiseti was the predominant species in soil samples from Nigeria and Zimbabwe, whileF. oxysporum was the predominant species recovered from soil from Lesotho.Contribution No. 1881, Fusarium Research Center, Department of Plant Pathology, The Pennsylvania State University.     

Biological control of Orobanche spp. with Phytomyza orobanchia, a review

This review summarises theavailable information on the biocontrol agentPhytomyza orobanchia Kalt. (Diptera, Agromyzidae). It gives an overview of a rareexample of weed biocontrol using insects in aninundative approach. A high diversity ofphytophagous insects has been collected onparasitic weeds of the genus Orobanche(Orobanchaceae). For biological control of Orobanche spp., only insects like P. orobanchia whose host range is restricted toOrobanche spp. are of interest. Of the140 Orobanche spp. described in total,P. orobanchia is reported from 21 species. The larvae of P. orobanchia minein Orobanche shoots and capsules. As aconsequence, a natural reduction of Orobancheseed production by 30 to almost 80%has been reported from different countries. Theefficacy of P. orobanchia under naturalconditions is limited by low temperatures, cultural practices and natural enemies. Tostrengthen the natural population and itsimpact, inundative releases of P. orobanchia adults at the beginning of Orobanche emergence have to be undertaken. Different methods for the application of P. orobanchia in biocontrol of Orobanche spp. have been developed in theformer Soviet Union. Releases of 500 to 1000adults/ha resulted in a reduction of up to 96%of the Orobanche seed production.However, due to the 10 to 15 year longevity ofOrobanche seeds, further infestationswill occur in the following cropping seasons.Consequently, releases of P. orobanchiahave to be repeated continuously over severalyears to reduce the infestation to a tolerablelevel.     

Spargelstangenuntersuchungen zur Haupterntezeit auf Infektionen mitFusarium spp. und Kontaminationen mit Fumonisin B1

Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization byFusarium spp., particularlyF. proliferatum. Potentially toxigenic fungi such asF. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B₁ inF. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection byF. proliferatum and otherFusarium spp. was highly dependent on location and sampling date. The dominantFusarium species among the endophytic microflora wasF. oxysporum. Other frequently isolated species includedF. proliferatum, F. sambucinum, F. culmorum, F. avenaceum andF. equiseti. The incidence ofF. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20–47% of the spears examined were infected withF. proliferatum. Further exploration of FB₁ generation in asparagus is required because the low levels of FB₁ (10–50 (μg/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.

     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

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.     

Effect of chitosan for the control of potato diseases caused by Fusarium species

The antifungal activity of chitosan against Fusarium spp. was investigated based on in vitro and in vivo assays, and its possible modes of action were also explored. Chitosan applied at 4.0 g/L of acetic acid-distilled water solution significantly decreased the mycelial growth of Fusarium oxysporum, Fusarium sambucinum and Fusarium graminearum by 88.4%, 89.0% and 89.8%, respectively. Tuber treatment by chitosan (4.0 g/L) of acetic acid-distilled water solution, prior to inoculation, reduced dry rot severity induced by F. oxysporum and F. sambucinum by 60.0% and 48.2%, respectively. When tested as plant treatment, potato plants inoculated with Fusarium species, exhibited 33.5%–45.3% less wilting severity as compared to the control. This abiotic treatment improved the phenolic compounds activities and defence-related enzymes such as peroxidase and polyphenoloxidase in potato tubers inoculated with Fusarium spp. Results clearly demonstrated that chitosan could be explored as an alternative agent to chemical fungicides for the control of tuber dry rot and Fusarium wilt through induction of the plant defence system.     

Frequency and diversity of Fusarium spp. colonizing roots of Egyptian cottons

Abstract

Fusarium species are known to play a role in several diseases of cotton including the seedling disease complex, wilt, and boll rot. Therefore, a mycoflora study was conducted in 1998 in order to identify Fusarium species found in association with cotton roots. A total of 109 samples of cotton seedlings infected with post-emergence damping-off or rotted roots of adult plants were obtained from different cotton-growing areas in Egypt. Forty-six isolates were recovered and were identified as follows: F. oxysporum (28 isolates), F. moniliforme (9), F. solani (6), F. avenaceum (2), F. chlamydosporum (1). F. oxysporum, F. moniliforme and F. solani, the dominant species, accounted for 60.9%, 19.6% and 13% of the total isolates, respectively in 1998. F. oxysporum showed the highest isolation frequency in Beharia and Minufiya while F. moniliforme showed the most isolation frequency in Minufiya and Gharbiya. F. oxysporum was one of the major taxa of the Fusarium assemblage from Giza 70. F. oxysporum showed the most frequently isolated fungus in May while F. moniliforme and F. solani were the most frequently isolated fungi in August. Isolation frequency of Fusarium spp. during July and August was significantly greater than that of April or June. This implies that cotton roots are subjected more to colonization by Fusarium spp. as plants mature. Regarding pathogenicity, of the 46 isolates of Fusarium spp. tested under greenhouse conditions, 38 isolates (82.4%) were pathogenic to seedlings of Giza 89. This study indicates that F. oxysporum and F. moniliforme are important pathogens in the etiology of cotton damping-off in Egypt.