Fluorescent Viability Stains to Probe the Metabolic Status of Aflatoxigenic Fungus in Dual Culture of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Pichia anomala</Emphasis>

The metabolic activity of the aflatoxigenic fungus, Aspergillus flavus co-cultured with the biocontrol yeast, Pichia anomala was examined using several viability stains. Both the FUN-1 stain and the combined use of DiBAC₄(5) with CDFA-AM stains were applied in this study. The results suggest that the ATP-generating system in A. flavus was inactivated as the ratio of yeasts to fungi increased in the dual culture. A decrease in hyphal membrane potential and esterase activity was substantiated by the combined stains of DiBAC₄(5) and CDFA-AM. Reduced metabolic function in conjunction with cell wall damage of A. flavus hindered the growth and biomass production of this fungus. Viability stains such as FUN-1 and DiBAC₄(5) with CDFA-AM may assist in elucidating the biocontrol mechanism by allowing for the visualization of the antagonistic effect of yeast species on target fungi in situ, as well as for screening potent biocontrol yeast agents against fungal pathogens.     

Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum

The objectives of this work were to assess the optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the biocontrol activity of non-adapted and acid-adapted yeasts in controlling apple blue mold caused by Penicillium expansum. R. paludigenum grown in malic and lactic acid treatments were stimulated after 12 h incubation. Moreover, medium modified with malic and lactic acid significantly enhanced the acid tolerance of R. paludigenum (p?<?0.05). In acid tolerance response test, the highest viability of R. paludigenum was obtained at initial pH of 5.5 in the NYDB medium modified with malic acid (91.6 %). In addition, all R. paludigenum treatments significantly reduced the disease incidences and lesion diameters of blue mold in apples. Furthermore, there was no significant difference between acid-adapted and unadapted yeasts in the apple wounds after 48 h dynamics. Acid stress improved R. paludigenum viability under acidic conditions. However, there was no significant difference between acid-adapted and unadapted yeasts in controlling P. expansum on apple fruit (p?<?0.05). These results indicate the potential for maintaining the survival level of biocontrol agents by physiological inducement strategy.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.     

Biochemical characterization and growth patterns of new yeast isolates

African sorghum opaque beers play a vital role in the diet of millions of consumers. In the current study we investigated the growth profiles of yeast strains isolated from kpete-kpete, a traditional starter used to produce tchoukoutou, an opaque sorghum beer in Benin. 10 yeast strains were isolated from sorghum beer starters and cultivated under both liquid and solid media for phenotypic growth characterization. All yeast isolates were able to grow both on solid and liquid media. Based on their growth profiles, the isolates were clustered into three groups: (i) the aggressive growth pattern (30 %), (ii) the moderate growth pattern (50 %), and (iii) the slow growth pattern (20 %). Based on gene expression pattern, absorbance (A_600nm) and diameter of growth in both liquid and solid media respectively, yeast strains YK34, YK15 and YK48 were clustered in the first group, and referred to as the most aggressive growth strains, followed by group 2 (YK24, YK5, YK12, YK20, YK2) and group 3 (YK37, YK41). This growth pattern was confirmed by Invertase gene expression profiling of the yeasts showing group 1 with high level of Invertase gene expression followed by group 2 and group 3 respectively. Our results suggest that YK34, YK15 and YK48 and YK2 yeast strains constitute the best candidates in fermentation of sorghum beer production based on growth rate and assimilation of carbon and nitrogen sources.     

Unraveling the decolourizing ability of yeast isolates from dye-polluted and virgin environments: an ecological and taxonomical overview

Microcosm assays with dye-amended culture media under a shot-feeding strategy allowed us to obtain 100 yeast isolates from the wastewater outfall channel of a dyeing textile factory in Tucumán (Argentina). Meanwhile, 63 yeast isolates were obtained from Phoebe porphyria (Laurel del monte) samples collected from Las Yungas rainforest (Tucumán), via a classical isolation scheme. Isolated yeasts, both from dye-polluted and virgin environments, were compared for their textile dye decolourization ability when cultured on solid and liquid media. Nine isolates from wastewater and 17 from Las Yungas showed the highest decolourization potential on agar plates containing six different reactive dyes, either alone or as a mixture. Five yeasts from each environment were further selected on the basis of their high dye removal rate in Vilmafix^® Red 7B-HE- or Vilmafix^® Blue RR-BB-amended liquid cultures. Yeasts from wastewater showed slightly higher decolourization percentages after 36 h of culture than yeasts from Las Yungas (98?C100% vs. 91?C95%, respectively). However, isolates from Las Yungas exhibited higher specific decolourization rates than isolates from effluents (1.8?C3.0 vs. 0.9?C1.3 mg g^?1h^?1, respectively). All selected isolates were first grouped according to microsatellite-PCR analysis and representative isolates from each group were subsequently identified based on the 26S rRNA gene sequence analysis. Yeasts from wastewater were identified as the ascomycetous Pichia kudriavzevii (100%) and closely related to Candida sorbophila (99.8%), whilst yeasts from Las Yungas were identified as the basidiomycetous Trichosporon akiyoshidainum and Trichosporon multisporum. It is suggested that findings concerning yeast selection during screening programs for dye-decolourizing yeasts may be explained in the light of the copiotroph-oligotroph microorganisms rationale.     

Osmotic stress adaptation,compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Vegetative compatibility and phenotypic characterization as a means of determining genetic diversity of Aspergillus flavus isolates

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2010, which resulted in 215 deaths. We examined the possible reasons for these frequent aflatoxicosis outbreaks in Kenya by studying Aspergillus flavus diversity, phenotypes and mycotoxin profiles across various agricultural regions. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu counties. Out of 37 isolates, nitrate non-utilizing auxotrophs complementation test revealed 20 vegetative compatibility groups. We designated these groups by the prefix “KVCG”, where “K” represented Kenya and consequently assigned numbers 1–20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L-, S- and S-/L-morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13), respectively. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC and HPLC analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. Diversity index (H′) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). To our knowledge, this is the first reported findings for A. flavus diversity and distribution in Nandi, Homa Bay and Kisumu counties and may assist current and future researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination as has been researched in Makueni and neighbouring counties.     

Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition

Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa.     

Predicted ecological niches and environmental resilience of different formulations of the biocontrol yeast <Emphasis Type="Italic">Candida sake</Emphasis> CPA-1 using the Bioscreen C

Environmental resilience of biocontrol microorganisms has been a major bottleneck in the development of effective formulations. Candida sake is an effective biocontrol agent (BCA) against Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer, and different formulations of the BCA have been optimised recently. The objective of this study was to compare the relative tolerance of different dry and liquid formulations of the biocontrol yeast C. sake CPA-1 to interacting environmental conditions using the Bioscreen C. Initially, the use of this automated turbidimetric method was optimised for use with different formulations of the biocontrol yeast. The best growth curves were obtained for the C. sake CPA-1 strain when grown in a synthetic grape juice medium under continuous shaking and with an initial concentration of 10⁵ CFUs ml^?1. All the formulations showed a direct relationship between optical density values and yeast concentrations. Temperature (15–30 °C) and water activity (a_w; 0.94–0.99) influenced the yeast resilience most profoundly, whereas the effect of pH (3–7) was minimal. In general, the liquid formulation grew faster in more interacting environmental conditions but only the yeast cells in the dry potato starch formulation could grow in some stress conditions. This rapid screening method can be used for effective identification of the resilience of different biocontrol formulations under interacting ecological abiotic conditions.     

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H₂O₂ and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H₂O₂. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H₂O₂. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H₂O₂, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.     

Effect of entomopathogenic Aspergillus strains against the pea aphid,Acyrthosiphon pisum (Hemiptera: Aphididae)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae, which is a well-known entomopathogen in the literature. Lethal concentrations (LC₅₀ and LC₉₀) were 1.23 × 10³ and 1.34 × 10⁷ spores/ml for A. flavus, 4.95 × 10² and 5.65 × 10⁷ spores/ml for A. clavatus, and 3.67 × 10³ and 9.71 × 10⁷ spores/ml for M. anisopliae 5 days after treatment. Mycelia development and sporulation on adult cadavers were observed 48 h after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains, suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also to our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids.     

Fungicide resistance of Botrytis cinerea in tomato greenhouses in the Canary Islands and effectiveness of non-chemical treatments against gray mold

Tomato greenhouses in the Canary Islands, Spain, were surveyed to estimate frequencies of resistance to benzimidazoles, dicarboximides, anilinopyrimidines and N-phenylcarbamates in Botrytis cinerea. Resistance to carbendazim, iprodione, pyrimethanil and diethofencarb was found in 74.2, 86.4, 28.8 and 31.8 % of isolates, respectively. Benzimidazole- and anilinopyrimide-resistant isolates were highly resistant, showing EC₅₀ values above 500 µg/ml carbendazim and a mean EC₅₀ value of 28.42 µg/ml pyrimethanil, respectively. By contrast, a low level of resistance was observed among dicarboximide-resistant isolates (mean EC₅₀ value of 1.81 µg/ml iprodione). Phenotypes with double resistance to carbendazim and iprodione, and triple resistance to carbendazim, iprodione and pyrimethanil were the most common, occurring in 36.4 and 28.8 % of isolates. The surveyed greenhouses had never been treated with fenhexamid and Signum? (pre-packed mixture of boscalid and pyraclostrobin), and baseline sensitivities of B. cinerea isolates to these fungicides were determined. The EC₅₀ values were within the range of 0.009–0.795 µg/ml fenhexamid and of 0.014–0.48 µg/ml Signum. In addition, available formulations based on elicitors of plant defense response and biocontrol agents were evaluated against B. cinerea in tomato plants under semi-controlled greenhouse conditions, the yeast Candida sake CPA-1 being able to reduce gray mold significantly when it was applied on petiole wounds and the plants were inoculated 24 h later. Likewise, C. sake was effective against B. cinerea in harvested tomato fruits, yeast-treated tomatoes showed a 70.66 and 30.31 % reduction in the diameters of decay lesions compared with controls after 10 days of storage at 20 and 9 °C, respectively.     

Aflatoxin Production by Aspergillus flavus as Related to Various Temperatures

Two aflatoxin-producing isolates of Aspergillus flavus were grown for 5 days on Wort media at 2, 7, 13, 18, 24, 29, 35, 41, 46, and 52 C. Maximal production of aflatoxins occurred at 24 C. Maximal growth of A. flavus isolates occurred at 29 and 35 C. The ratio of the production of aflatoxin B₁ to aflatoxin G₁ varied with temperature. Aflatoxin production was not related to growth rate of A. flavus; one isolate at 41 C, at almost maximal growth of A. flavus, produced no aflatoxins. At 5 days, no aflatoxins were produced at temperatures lower than 18 C or higher than 35 C. Color of CHCl₃ extracts appeared to be directly correlated with aflatoxin concentrations. A. flavus isolates grown at 2, 7, and 41 C for 12 weeks produced no aflatoxins. At 13 C, both isolates produced aflatoxins in 3 weeks, and one isolate produced increasing amounts with time. The second isolate produced increasing amounts through 6 weeks, but at 12 weeks smaller amounts of aflatoxins were recovered than at 6 weeks.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Staurosporine from the endophytic Streptomyces sp. strain CNS-42 acts as a potential biocontrol agent and growth elicitor in cucumber

Chinese medicinal plants and their surrounding rhizospheric soil serve as promising sources of actinobacteria. A total of 180 actinobacteria strains were isolated from the rhizosphere soil, leaves, stems, and roots of nine selected plants and have been identified as potential biocontrol agents against Fusarium oxysporum f. sp. cucumerinum. An endophytic strain CNS-42 isolated from Alisma orientale showed the largest zone of inhibition demonstrating a potent effect against F. oxysporum f. sp. cucumerinum and a broad antimicrobial activity against bacteria, yeasts, and other pathogenic fungi. The in vivo biocontrol assays showed that the disease severity index was significantly reduced (P < 0.05), and plant shoot fresh weight and height increased greatly (P < 0.05) in plantlets treated with strain CNS-42 compared to the negative control. This isolate was identified as Streptomyces sp. based on cultural, physiological, morphological characteristics, and 16S rRNA gene analysis. Further bioassay-guided isolation and purification revealed that staurosporine was responsible for its antifungal and plant growth promoting activities and the latter property of staurosporine is reported for the first time. The in vivo assay was further performed and indicated that staurosporine showed good growth promoting effect on the plant shoot biomass of cucumber. This is the first critical evidence identifying CNS-42 as a biocontrol agent for the soil borne pathogen, F. oxysporum f. sp. cucumerinum.     

Molecular characterisation and biological control of Aspergillus flavus isolates from Saudi Arabia

Abstract

Aspergillus flavus is a phytopathogenic fungus that produces toxic compounds, aflatoxins, in infected plant tissues which harm human and animal health. In this study, 57 food/feed products were collected from 9 locations in Aseer, KSA. A total of 93 isolates were recovered from the samples and were identified as Aspergillus spp. based on their cultural and microscopic characteristics. Six isolates (Af3, Af23, Af24, Af26, Af45 and Af48) were selected and confirmed as A. flavus using polymerase chain reaction (PCR), sequencing of ITS1-5.8s-ITS2 rDNA region and phylogenetic analyses. The six sequences were deposited in the GenBank under the accession numbers of KU561932, KU561934, KU561935, KU561936, KU561937 and KU561938, respectively. Random amplification of polymorphic DNA (RAPD-PCR) of the six isolates using five primers (OPA-2, OPA-3, OPA-9, OPA-11 and OPA-15), produced polymorphic DNA bands of 12, 36, 25, 1 and 1, respectively. The band sizes ranged from 130 to 1600?bp, whereas no monomorphic bands were observed. The bio-control of the six selected A. flavus isolates using three locally isolated yeasts (Candida davisiana, Rhodotorula graminis and Exophiala dermatitidis) was assessed. On solid media, the three yeast strains inhibited all tested A. flavus isolates. The most effective yeast strain was R. graminis. In liquid media, both yeast strains C. davisiana and R. graminis inhibited the dry weights of the six A. flavus isolates. Bio-control approaches of A. flavus could help controlling the pathogen, ultimately, reduce the risk of aflatoxins in human and animal supplies and reduce the use of chemicals that affect the environment and health.     

Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach

Endophytic microorganisms inhabit internal plant tissues in the host plant without causing any symptoms or negative effects. Although the diversity of endophytes has been evaluated by both culture-dependent and culture-independent methods, less information is available on yeast communities. Therefore, in this study a culture-independent method was used to examine endophytic yeasts associated with rice leaves based on the large subunit of ribosomal DNA using a semi-nested PCR technique. Sequence analysis indicated that the colonization frequency and the relative species frequency (RF) of endophytic yeast phylotypes were 0.41 and 0.06, respectively, and the majority of the yeast phylotypes were basidiomycetous yeasts. The phylotypes were designated as five known species (Cryptococcus victoriae, Debaryomyces hansenii, Debaryomyces vindobonensis, Meyerozyma guilliermondii and Pseudozyma antarctica), together with seventeen phylotypes closest to Candida metapsilosis, Cryp. foliicola, Cryp. laurentii, Pseudozyma abaconensis, Pseudozyma aphidis and Trichosporon asahii, among which some could be novel species. The most prevalent phylotypes were those closest to Cryp. foliicola (47.5 % RF) followed by D. hansenii (22.8 % RF) and P. antarctica (16.8 % RF). The presence of the phylotypes related to species known for their potential applications as biocontrol agents and plant growth promoting hormone producers suggests that they may have valuable applications. In addition, our findings revealed the occurrence of novel phylotypes at high frequency, which should encourage extensive studies to discover novel yeast species and to understand their roles in the rice leaves.