Selection of non-toxigenic strains of Aspergillus flavus for biocontrol of aflatoxins in maize in Thailand

Aflatoxin production in maize and peanuts remains a major public health problem, especially in developing countries. One promising method for combating aflatoxin formation is biocontrol using competitive exclusion, a management strategy currently being studied in maize crops in Thailand. It is important that the strains of Aspergillus flavus used in biocontrol be non-toxigenic and be incapable of reversion to toxigenicity. In the current study, 80 non-toxigenic strains of A. flavus, randomly selected from commercially produced dried maize samples from several sources in Thailand, were screened for their potential as biocontrol strains by examining the 24 aflatoxin biosynthesis genes, using a PCR assay. Assessment of the presence or absence of PCR products provides an indication of the function of pathway genes. Of the 80 strains, 78 showed no PCR products from one or more genes in the aflatoxin biosynthesis pathway. Twenty-three isolates showed only one failure, in the aflD (nor-1) gene, but most isolates failed to produce a PCR product for two or more genes. Nineteen isolates (24%) failed to show a PCR product in 10 or more genes. Altogether, 45 PCR product patterns were observed, usually common to only one or two isolates, indicating great diversity in the aflatoxin biosynthesis pathway in A. flavus isolates taken from dried Thai maize. Although the absence of a particular PCR product is not conclusive evidence that the particular gene is non-functional, the absence of several such PCR products provides reasonable evidence that the isolate in question will not revert to toxigenicity in the field.     

A new method for in-situ monitoring of the underground development of Orobanche cumana in sunflower (Helianthus annuus) with a mini-rhizotron

AIMS: To develop an in-situ, non-destructive method for observation and monitoring of the underground developmental stages of the root parasite Orobanche cumana. SCOPE: The parasitic weed Orobanche causes severe damage to vegetables and field crops. Most of the damage caused to the crops occurs during the underground, unobservable parasitism stage. Sunflower (Helianthus annuus 'Adi') plants were planted in soil that was artificially inoculated with O. cumana seeds. Clear Plexiglas mini-rhizotron plastic observation tubes were inserted into the soil. Seed germination, early stage of penetration, and formation of tubercles and spikes were observed non-destructively and were monitored throughout the growing season by mean of a mini-rhizotron camera. Use of this technology enabled the complete individual parasite life cycle from the very early development (including germination) to Orobanche shoot to be monitored. In addition, the effect of the systemic herbicide Cadre (imazapic) on the development of O. cumana was inspected and quantified. CONCLUSIONS: This novel methodology facilitates the in-situ study of major aspects of the host-parasite interaction and of parasite suppression, such as parasitism dynamics, parasite growth rate, and the effect of chemical treatments on the parasite.     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

In vitro studies on the potential for biological control on Aspergillus section Flavi by Kluyveromyces spp

AIMS: Antagonist activity of Kluyveromyces spp. isolates on Aspergillus section Flavi was studied. METHODS AND RESULTS: The screening of isolates were made through studies of growth at different water activities and temperatures, index of dominance (I(D)), ecological similarity, antifungal activity and impact on aflatoxin B1 accumulation. High optical density was obtained at 25 and 30 degrees C and 48 h of incubation. Cell growth decreases with decrease in water activity. The predominant interaction was mutual intermingling at a(w) = 0.982 and 0.955, while at a(w) = 0.999 and 0.937 mutual inhibition for contact was exhibited. All isolates were catabolically identical to Aspergillus section Flavi and compete by nutritional source. At high water activities yeasts showed inhibitory activity on Aspergillus strains, inhibition percentages varied between 75 and 100%. The isolates Y9, Y14, Y16, Y22, Y25 and Y33 showed antifungal activity and inhibitory activity on aflatoxin B1 accumulation at all water activities assayed from all Aspergillus section Flavi strains. CONCLUSIONS: The data show that the isolates selected in a wide range of environmental conditions could exert their roll like biological control agents for Aspergillus section Flavi in storage maize ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolates of Kluyveromyces spp. may have practical value in the postharvest control of storage maize.     

Use of microsatellite markers to assess the competitive ability of nontoxigenic Aspergillus flavus strains in studies on biocontrol of aflatoxins in maize in Thailand

Many nontoxigenic strains of Aspergillus flavus have been used in studies on biocontrol by competitive exclusion, but assessing their competitive ability is difficult. This paper reports on the use of a microsatellite marker technique for assessing competitiveness. The chosen microsatellite markers were able to differentiate, at an individual level, between the four biocontrol strains used in a study on the biocontrol of aflatoxins in maize in Thailand. The microsatellite markers were then used to determine which of the four biocontrol strains used were identical with 86 nontoxigenic strains of A. flavus taken from dried maize samples produced in that study. Fifty-one of the 86 strains (59%) were identified as one of the four biocontrol strains, with another four likely to be so. Analysis of microsatellites in A. flavus strains taken from dried samples at the conclusion of a field trial was shown to be of value in understanding the competitive ability of the specific strains used for biocontrol. This method provides an objective assessment of the competitiveness of biocontrol strains.     

A strain of Aspergillus flavus from China shows potential as a biocontrol agent for aflatoxin contamination

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Strains of A. flavus that are non-aflatoxigenic (i.e., incapable of secreting aflatoxins) have proven effective in controlling contamination by these aflatoxin producing species in the field. In the present study, a non-aflatoxigenic A. flavus strain, GD-3, was isolated from a peanut field in Guangdong Province, China. Polymerase chain reaction (PCR) analysis showed that 12 aflatoxin biosynthesis genes (aflT, pksA, nor-1, fas-2, fas-1, aflR, aflJ, adhA, estA, norA, ver-1 and verA) were deleted in GD-3. Co-inoculation with a toxigenic strain, GD-15, at the ratio of 1:10, 1:1 or 10:1 (GD-3:GD-15), showed that GD-3 was capable of reducing detectable aflatoxin levels on three different substrates. This reduction ranged from 33% to 99% and correlated with competitor ratio. These results demonstrated that GD-3 was successful at reducing aflatoxin contamination and showed promise as a potential agent of biocontrol for local farmers.     

Infection of tubercles of the parasitic weed Orobanche aegyptiaca by mycoherbicidal Fusarium species

Progression of the infection by host-specific strains of Fusarium oxysporum and Fusarium arthrosporioides of Orobanche aegyptiaca (Egyptian broomrape) tubercles attached to tomato roots was tracked using light, confocal and electron microscopy. Mycelia transformed with the gene for green fluorescent protein were viewed using a confocal microscope. Fungal penetration was preceded by a rapid loss of starch, with approx. 10 % remaining at 9 h and no measurable starch at 24 h. Penetration into the Orobanche tubercles began by 12 h after inoculation. Hyphae penetrated the outer six cell layers by 24 h, reaching the centre of the tubercles by 48 h and infecting nearly all cells by 72 h. Most of the infected tubercles were dead by 96 h. Breakdown of cell walls and the disintegration of cytoplasm in and around the infected cells occurred between 48 and 96 h. Lignin-like material increased in tubercle cells of infected tissues over time, but did not appear to be effective in limiting fungal penetration or spread. Callose, suberin, constitutive toxins and phytoalexins were not detected in infected tubercles, suggesting that there are no obvious defence mechanisms to overcome. Both Fusarium spp. pathogenic on Orobanche produced fumonisin-like ceramide synthase inhibitors, while fusaric acid was produced only by F. oxysporum in liquid culture. The organisms do not have sufficient virulence for field use (based on glasshouse testing), suggesting that virulence should be transgenically enhanced or additional isolates sought.     

Evaluation of bacteria and Trichoderma for biocontrol of pre-harvest seed infection by Aspergillus flavus in groundnut

Pre- and post harvest aflatoxin contamination of groundnut caused by Aspergillus flavus is a major problem in the semi-arid tropics. Fluorescent Pseudomonas, Bacillus and Trichoderma spp. potentially antagonistic to A. flavus were isolated from the geocarposphere (pod-zone) of groundnut and used successfully for the control of pre-harvest groundnut seed infection by A. flavus. In greenhouse and field experiments, inoculation of selected antagonistic strains on groundnut resulted in significant reduction of seed infection by A. flavus, and it also reduced >50% of the A. flavus populations (as cfu) in the geocarposphere of groundnut.     

Development of strain specific molecular markers for the Sclerotinia minor bioherbicide strain IMI 344141

The plant pathogenic fungus, Sclerotinia minor IMI 344141, has been developed as a bioherbicide for broadleaf weed control in turfgrass and a means to differentiate this biocontrol agent from like organisms is required. A strain specific molecular marker was developed to detect and monitor the Sclerotinia minor IMI 344141 bioherbicide strain. The method was based on polymerase chain reaction (PCR) amplification of two sequence-characterized amplified regions (SCAR) primer pairs for a first round PCR, and another two sets of nested primers was used for a second round PCR if higher sensitivity was needed. Sclerotinia minor IMI 344141 was successfully traced from both pure cultures and environmental samples originating from bioherbicide-released field trials. DNA of the S. minor bioherbicide isolate IMI 344141 was detected in the soil 2 months after application, but was not detected in the 3- and 9-month samples after application. When applied as a bioherbicide, S. minor (IMI 344141) did not persist into the following spring season in turf environments. This molecular detection method provides a mechanism to distinguish this isolate from related organisms and a tool to monitor behavior of the biocontrol agent S. minor IMI 344141 in nature, particularly in soil.     

Allelopathy of rice (Oryza sativa L.) root exudates and its relations with Orobanche cumana Wallr. and Orobanche minor Sm. germination

Allelopathy has been considered not only as an environmentally friendly approach for weed control but also a potential reason causing autotoxicity in crop production. In this study, the responses of seeds of lettuce, wheat, rice, clover broomrape (CB), and sunflower broomrape (SB) to the root exudates of rice cultivars were studied. Lettuce germination was promoted by root exudates of Yliangyou 3218 and I-Kung-Pao. Wheat seedling growth was inhibited by all nine rice species. I-Kung-Pao and Ganxin 203 exerted greater autotoxicity than other cultivars. Yongyou 15 and I-Kung-Pao induced the highest germination rate of CB, while Yongyou 13, Zhongzao 22, and I-Kung-Pao induced the highest germination rate of SB. A significantly correlation was noted between germination-inducing ability on broomrape seeds and allelopathic effects on target plants. It is suggested that using broomrape seeds germination is a better receptor for the identification of rice allelopathic potential.     

Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.     

Two granular formulations of Fusarium oxysporum f.sp. orthoceras to mitigate sunflower broomrape Orobanche cumana

Fusarium oxysporum Schlecht. f.sp. orthoceras (Appel & Wollenw.) Bilai, a potential biocontrol agent against Orobanche cumana Wallr.,was formulated into two granular forms, wheatflour kaolin (`Pesta') granules and sodium alginatepellets. The formulations were compared in terms ofeffectiveness for mitigating O. cumanaparasitism in sunflower and shelf-life forstorage. `Pesta' granules reduced the emergence of O. cumana shoots by 64% while sodium alginatepellets did not reduce the emergence rate but increased thepercentage of diseased O. cumana plants.Calculated efficacy of the application was better for`Pesta' granules. Viability of the formulatedmaterial tested in the laboratory was higher in sodium alginatepellets than in the `Pesta' formulation.However, a loss of virulence after six months of storage wasalso observed in sodium alginate pellets in agreenhouse experiment.     

Taxonomy of the Chinese Orobanche and Its Relationships with Related Genera

The morphological characters in the genus Orobanche were evaluated from the taxonomic point of view. The author finds that the plants of this genus are relatively similar to each other in respect to characters of vegetative organs, fruits and seeds. But the differences in the floral structures can be served as a basis for delimitating infrageneric taxa. The seed coat of 18 species and pollen grains of 6 species were also examined under scanning electron microscope (SEM). They seem to have little significance for distinguishing species. The result supports G. Beck’s (1930) division of the genus Orobanche into 4 sections, of which 2 occur in China, based on the characters of the inflorescence, bracteoles and calyx. The author considers that some characters, such as anther hairy or not, upper lip of corolla entire or not, lower lip longer or shorter than the upper one, the state of corolla-tube inflec tion and the hair type of filaments and plants, are important in distinguishing Chinese species. A key to the species of Orobanche in China is given. This genus consists of about 100 species, and is mostly confined to Eurasia, with over 60 species found in Caucasus and Middle Asia of USSR, where may be the mordern distribu tional centre. Orobanche L. in China is represented by 23 species, 3 varieties and l forma. As shown in Table 1, most species (12 species) are found in Xinjiang, which clearly shows a close floristic relationship between this region and Middle Asia of USSR. 6 species are endemic to China, of which 4 are confined to the Hengduan Mountains (Yangtze-Mekong-Salwin divide). The relationships between this genus and related ones of Orobanchaceae are also discussed. The author holds the following opinions: the genus Phelypaea Desf. should be considered as a member of Orobanche L. Sect. Gymnocaulis G. Beck, the monotypic genus, Necranthus A. Gilli endemic to Turkey, is allied with Orobanche L. Sect. Orobanche, the monotypic genus, Platypholis Maxim, endemic to Bonin Is. of Japan, is far from Orobanche L. in relation and should be regarded as a separate genus. The 11 OTU’s, including all the sections of Orobanche L. and 7 genera of Orobanchaceae, and 15 morphological characters were used in the numerical taxonomic treatment to test the above-mentioned suggestions. After standardization of characters, the correlation matrices were computerized. The correlation matrices were made to test the various clustering methods. At last the UPGMA clustering method was chosen and its result is shown in a phenogram. The result of numerical analysis is basically in accordance with the suggestions.     

Biocontrol of broomrape (Orobanche crenata Forsk. and Orobanche foetida Poir. ) by Pseudomonas fluorescens isolate Bf7-9 from the faba bean rhizosphere

Bacteria of the faba bean (Vicia faba L.)/Orobanche spp. root environment were evaluated for their potential use as biocontrol agents for the parasitic weed. Bacteria were isolated mainly from the rhizosphere of faba bean as well as from diseased Orobanche underground structures and an Orobanche-suppressive soil from three districts of northern Tunisia. Out of 351 bacterial isolates, 337 were tested for pathogenicity in an inverted pyramidal-shape screening programme including a Lactuca sativa L. seedlings bioassay, root-chamber and pot experiments. In pre-selection screening on L. sativa seedlings, 37 isolates (11%) showed a strong growth inhibitory effect, of which 70 and 84% also had a significant suppressive activity on the pre-emergence structures of O. foetida and O. crenata, respectively, in root-chamber experiments. Among five bacterial isolates selected for pot trials, strain Bf7-9 of Pseudomonas fluorescens showed high biocontrol activity against both species of Orobanche and positively influenced faba bean growth. The bacterium reduced shoot emergence of O. crenata and O. foetida by 64 and 76% and their dry weight by 39 and 63%, respectively, compared with non-inoculated controls. Pseudomonas marginalis strain Nc1-2 exhibited also a tendency to reduce incidence of O. crenata and to improve faba bean performance. Results of the present study suggest that application of naturally occurring rhizosphere bacteria offers an additional approach for biocontrol of Orobanche spp. that can supplement current methods of control in an integrated weed management strategy.     

致病性曲霉的耐药性研究进展

随着抗真菌药物在临床上的广泛使用,致病性真菌的耐药率越来越高,耐药曲霉对侵袭性曲霉病的诊治产生了重要影响。目前,致病性曲霉耐药性的确定主要依靠抗真菌药敏试验和分子诊断。在有关曲霉耐药机制的研究中,报道最多的是曲霉对唑类药物的耐药,其机制主要包括外排泵表达增加、靶酶Cyp51突变和表达水平增高、形成生物膜,以及热休克蛋白90（Hsp90）介导的信号通路参与而导致的耐药。本文就上述领域近年来的主要进展进行综述。     

Generation of transgenic asulam-resistant potatoes to facilitate eradication of parasitic broomrapes (Orobanche spp.), with the sul gene as the selectable marker

 Root-attaching parasitic flowering broomrapes (Orobanche spp.) are major constraints to vegetable, legume and sunflower production around the Mediterranean and elsewhere, with banned methyl-bromide fumigation or land abandonment of these affected crops as the major “solutions” to the problem. We report the specific generation of transgenic asulam-resistant potatoes as a way to eradicate this pest. The target-site resistance in the crop allows the herbicide to be translocated from treated leaves to the parasite via crop roots. This inhibitor of dihydropteroate synthase lethally then prevents folic acid biosynthesis in the parasite. Additionally, we demonstrate that asulam can be used directly in selecting resistant transformants, without the need for another selectable marker. Received: 18 June 1997 / Accepted: 17 July 1997     

Pollen morphology of the genus Orobanche L. (Orobanchaceae)

A comparative palynological study of 25 taxa of the genus Orobanche L. belonging to section Trionychon Wallr. and section Orobanche L. was carried out. A clear difference between the pollen grains of section Trionychon and section Orobanche was observed. The grains of section Trionychon are typically tricolpate, radially symmetrical, more or less oblate spheroidal, with a microreticulate, or scabrate-perforate sculpture. Section Orobanche on the other hand, had spheroidal, more or less inaperturate pollen grains, although some were tricolpate, but without any clear symmetry. The basic surface sculpture of the pollen in section Orobanche was scabrate to rugulate-perforate. Palynoiogicaliy, six groups can be recognized amongst the species of the genus Orobanche , lor which a key is provided.     

Evaluation of Streptomyces spp. and Bacillus spp. for biocontrol of Fusarium wilt in chickpea (Cicer arietinum L.)

A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.