Biocontrol Potential of Trichoderma Ghanense and Trichoderma Citrinoviride toward Pythium aphanidermatum.

Pythium-induced damping-off of cucumber is a major constraint to cucumber production in different parts of the world. Although chemical fungicides are used for managing this disease, they have many drawbacks to the environment. The ability of the antagonistic fungi isolated from the rhizosphere and endosphere of Dactyloctenium robecchii and Moraea sisyrinchium in the control of soilborne pathogen Pythium aphanidermatum was inspected. Native Trichoderma isolates, Trichoderma ghanense and Trichoderma citrinoviride, were isolated from plant stem and soil samples collected from Al-Seeb, Oman. Using a dual culture technique, the antagonistic activity of the fungal isolates against P. aphanidermatum was examined in vitro. Among Trichoderma isolates, T. ghanense was more efficient in restraining the mycelial growth of P. aphanidermatum, causing an inhibition percentage of 44.6%. Further, T. citrinoviride induced significantly lower cessation of P. aphanidermatum mycelial growth (31.3%). Microscopic and electrolyte leakage inspection of the pathogen mycelia depicted extreme morphological malformations in their mycelium, which can be attributed to the antifungal metabolites of antagonists. Greenhouse studies demonstrated the effectivity of T. ghanense in controlling Pythium damping-off of cucumber plants, where the number of surviving plants was over 90% when the biocontrol agents were used compared to 0 in the control plants. Furthermore, treatment of the plants with the antagonists promoted growth characteristics of plants compared to uninoculated plants. This included improvements in shoot and root lengths, leaf length and width, and dry weight. These findings suggest that T. ghanense and T. citrinoviride can be developed as alternatives to synthetic chemical fungicides to manage soilborne pathogens of cucumber. This research is also the first to clarify the biocontrol ability of T. citrinoviride and T. ghanense against cucumber damping-off caused by P. aphanidermatum.

Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman, based on morphology and molecular phylogeny.

Hymenagaricus has small to medium-sized mushrooms and the cap surface with squamulose pellicles, consisting of hymeniform or pseudoparenchymatous cells and yellowish-brown basidiospores. The species of Hymenagaricus are very similar to those of Xanthagaricus and it is extremely difficult to differentiate the species of both genera in the field. However, phylogenetically, both the genera are clearly distinct. In this study, we describe two new species of Hymenagaricus, i.e. H.wadijarzeezicus and H.parvulus from the southern part of Oman. Species descriptions are based on a combination of morphological characteristics of basidiomata and phylogenetic analyses of three gene regions: internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), the large subunit of nuclear ribosomal DNA (28S) and translation elongation factor one alpha (EF-1α). Full descriptions, micrographs and illustration of anatomical features, basidiomata photos and phylogenetic analyses results of the new taxa are provided. Morphological comparisons of new taxa with similar species and a key to species included in the phylogenetic analyses are also provided.

Plant-Disease-Suppressive and Growth-Promoting Activities of Endophytic and Rhizobacterial Isolates Associated with Citrullus colocynthis.

This study was conducted to investigate the antagonistic potential of endophytic and rhizospheric bacterial isolates obtained from Citrullus colocynthis in suppressing Fusarium solani and Pythium aphanidermatum and promoting the growth of cucumber. Molecular identification of bacterial strains associated with C. colocynthis confirmed that these strains belong to the Achromobacter, Pantoea, Pseudomonas, Rhizobium, Sphingobacterium, Bacillus, Sinorhizobium, Staphylococcus, Cupriavidus, and Exiguobacterium genera. A dual culture assay showed that nine of the bacterial strains exhibited antifungal activity, four of which were effective against both pathogens. Strains B27 (Pantoea dispersa) and B28 (Exiguobacterium indicum) caused the highest percentage of inhibition towards F. solani (48.5% and 48.1%, respectively). P. aphanidermatum growth was impeded by the B21 (Bacillus cereus, 44.7%) and B28 (Exiguobacterium indicum, 51.1%) strains. Scanning electron microscopy showed that the strains caused abnormality in phytopathogens' mycelia. All of the selected bacterial strains showed good IAA production (>500 ppm). A paper towel experiment demonstrated that these strains improved the seed germination, root/shoot growth, and vigor index of cucumber seedlings. Our findings suggest that the bacterial strains from C. colocynthis are suppressive to F. solani and P. aphanidermatum and can promote cucumber growth. This appears to be the first study to report the efficacy of these bacterial strains from C. colocynthis against F. solani and P. aphanidermatum.

Relationship between Dubas Bug (Ommatissus lybicus) Infestation and the Development of Fungal-Induced Leaf Spots in Date Palms (Phoenix dactylifera).

The dubas bug (Ommatissus lybicus) (Hemiptera: Tropiduchidae) is a serious pest in date palms in several date-producing countries, including Oman. Infestation results in a severe reduction in yield and a weakening of date palm growth. In addition, egg laying, which causes injuries to date palm leaves, results in the development of necrotic lesions on the leaves. This study aimed at investigating the role of fungi in the development of necrotic leaf spots following dubas bug infestation. Leaf samples developing leaf spot symptoms were collected from dubas-bug-infested leaves, as the leaf spot symptoms were not observed on the non-infested leaves. Isolation from date palm leaves collected from 52 different farms yielded 74 fungal isolates. Molecular identification of the isolates revealed that they belonged to 31 fungal species, 16 genera, and 10 families. Among the isolated fungi, there were five Alternaria species, four species each of Penicillium and Fusarium, three species each of Cladosporium and Phaeoacremonium, and two species each of Quambalaria and Trichoderma. Out of the thirty-one fungal species, nine were pathogenic on date palm leaves and induced varying levels of leaf spot symptoms. The pathogenic species were Alternaria destruens, Fusarium fujikuroi species complex, F. humuli, F. microconidium, Cladosporium pseudochalastosporoides, C. endophyticum, Quambalaria cyanescens, Phaeoacremonium krajdenii, and P. venezuelense, which were reported for the first time as leaf spot causal agents in date palms. The study provided novel information on the effect of dubas bug infestation in date palms on the development of fungal infection and associated leaf spot symptoms.

Effectiveness of Endophytic and Rhizospheric Bacteria from Moringa spp. in Controlling Pythium aphanidermatum Damping-Off of Cabbage.

In this study, endophytic and rhizospheric bacteria were isolated from Moringa olifera and M. perigreina from Oman, and their in vitro antagonistic activity against Pythium aphanidermatum was tested using a dual culture assay. The promising strains were tested further for their compatibility and potential for plant growth promotion, biofilm formation, antifungal volatile organic compound (VOC) production, and the biological control of P. aphanidermatum damping-off of cabbage (Brassica oleracea L.) under greenhouse conditions. A total of 12 endophytic and 27 rhizospheric bacteria were isolated from Moringa spp. Among them, Bacillus pumilus MPE1 showed the maximum antagonistic activity against P. aphanidermatum in the dual culture assay, followed by Paenibacillus glucanolyticus MPE3 and Pseudomonas indica MOR3 and MOR8. These bacterial isolates induced abundant morphological abnormalities in the hyphae of P. aphanidermatum, as observed via scanning electron microscopy. The in vitro cross-streak assay showed that these bacterial isolates were compatible among themselves, except for P. indica MOR8 × P. glucanolyticus MPE3. These antagonists released VOCs that restricted the growth of P. aphanidermatum in an in vitro assay. These antagonistic bacteria released 2,4-dimethylheptane and 4-methyloctane as the predominant volatile compounds. Of the four antagonistic bacterial strains, P. indica MOR8 was capable of forming biofilm, which is considered a trait that improves the efficacy of rhizosphere inoculants. The results of the greenhouse experiments showed that the soil treatment with B. pumilus MPE1 showed the highest reduction (59%) in the incidence of P. aphanidermatum damping-off in cabbage, evidencing its potential as a biological control agent for the management of this disease. Further research is needed to characterize the antifungal traits and activities of B. pumilus MPE1 and to assert its potential use against other soil-borne plant pathogens.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus.

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

The Genus Xanthagaricus: An Updated Global Species Distribution and Phylogeny with the Description of Two New Species from Oman.

Xanthagaricus is a saprotrophic mushroom genus with small-sized basidiomata in the family Agaricaceae (Agaricales). Prior to this study, 26 species belonging to this genus have been described and published. In this study, we reported Xanthagaricus for the first time from Oman with the description of two new species. Basidiomata of the new species Xanthagaricus appendiculatus and X. omanicus were collected during the monsoon rains of summer in 2018 in the southern coastal region of Oman. Species descriptions are based on morphological and molecular characterization. Phylogenetic analyses based on internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS) of the nuclear ribosomal DNA clustered the new species in the Xanthagaricus clade with strong statistical support. The new species Xanthagaricusappendiculatus can be distinguished from other species by its purplish pileus with umbonate disc and X. omanicus with the largest pileus (70-90 mm diameter) among the known species of the genus. A detailed description, photographs, line drawings, and a phylogenetic tree showing the position of both new species are provided. A dichotomous key to the known taxa of Xanthagaricus is proposed. Morphological comparisons of new species with known Xanthagaricus taxa are provided. Our observations highlight the diversity of Xanthagaricus and other lepiotaceous mushrooms in southern Oman and further document the need for additional systematic focus on the region's fungi.

In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus.

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.

Antagonistic Activity of Endophytic and Rhizosphere Fungi Isolated From Sea Purslane (Sesuvium portulacastrum) Against Pythium Damping off of Cucumber.

This study was conducted to investigate the antagonistic activity of endophytic and rhizosphere fungi isolated from a medicinal plant, Sesuvium portulacastrum, against Pythium aphanidermatum, the cause of damping off of cucumber. A total of 40 endophytic and 19 rhizosphere fungi were isolated from S. portulacastrum. Three endophytic isolates and two rhizosphere isolates gave >50% suppression of P. aphanidermatum in the in vitro dual-culture tests. Scanning electron microscopic studies at the inhibition zone showed hyphae wall damage and abnormal mycelial growth of the genus Pythium. Molecular analysis identified the antagonistic endophytes as Aspergillus insulicola (isolate A435), A. insulicola (A419), and Aspergillus melleus (A412) and the rhizosphere antagonists as Aspergillus terreus (A213) and Aspergillus luchuensis (A116). Except for A116, the culture filtrates of the other antagonists significantly increased the electrolyte leakage from Pythium mycelia, whereas ethyl acetate extracts of A435, A412, and A213 showed significant growth suppression. All five antagonists were able to produce varying amounts of cellulase and ß-glucanase enzymes. However, A435, A412, and A213 showed significantly higher cellulase activity, whereas A435 and A116 showed the highest ß-glucanase activity. Controlled glasshouse growth experiments showed that isolates A435 and A116 resulted in up to 70% control of damping off, whereas isolates A412 and A213 showed 30 to 40% damping-off control. The antagonists A435, A116, and A213 also contributed to increased cucumber shoot length as well as shoot and root dry mass. The synergetic effects of metabolites and hydrolytic enzymes could be the reason for the variation between isolates in the antagonistic activity and cucumber growth promotion. This study reports for the first time A. insulicola, A. melleus, and A. luchuensis as potential biocontrol agents against P. aphanidermatum-induced damping off of cucumber.

Role of Adhatoda vasica (L.) Nees leaf extract in the prevention of aflatoxin-induced toxicity in Wistar rats.

BACKGROUND: Aflatoxin contamination of various foodstuffs and agricultural commodities is a major problem worldwide. Several strategies have been reported for the detoxification of aflatoxins in contaminated foods and feeds, but all these methods have their own shortcomings. Traditional medicinal plants are potential sources of aflatoxin-detoxifying compounds. In this study a spray-dried formulation of Adhatoda vasica (L.) Nees leaf extract was prepared and its chemopreventive effect on aflatoxin B1 (AFB1)-induced biochemical changes in the liver and serum of Wistar rats was investigated. RESULTS: Administration of AFB1 (1.5 mg kg(-1) body weight (BW) intraperitoneally) to rats significantly reduced the activities of superoxide dismutase and catalase in liver tissues and increased the activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase and the levels of very-low-density lipoprotein, low-density lipoprotein and cholesterol in blood serum. However, pre-feeding of rats with A. vasica formulation (500 mg kg(-1) BW for 7 days) protected the animals from AFB1-induced biochemical changes during subsequent exposure to AFB1. CONCLUSION: Pre-feeding of rats with A. vasica formulation counteracted the hepatic dysfunction induced by subsequent treatment with AFB1. This formulated A. vasica extract offers a biologically safe alternative to detoxify aflatoxin and has huge potential to be used in the poultry industry to reduce aflatoxicosis.

Induction of thaumatin-like proteins (TLPs) in Rhizoctonia solani-infected rice and characterization of two new cDNA clones.

Thaumatin-like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani. Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non-pathogenic bacterium, Pseudomonas syringae pv. syringae. By probing a cDNA expression library prepared from RNA isolated from R. solani-infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1-kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.