Isolation and identification of pathogenic fungi and oomycetes associated with beans and cowpea root diseases in Oman

The purpose of this study was to characterize fungal and oomycete species associated with root diseases of Phaseolus vulgaris, Vigna unguiculata, V. radiata and Vicia faba in Oman. Root samples were collected from plants suffering from weakened growth and yellowing symptoms. Fungal species were isolated on 2.5% potato dextrose agar amended with 10 mg l−1 rifampicin and 200 mg l−1 of ampicillin and identification was based on sequences of the internal transcribed spacer region of the ribosomal RNA gene (ITS rRNA), glycerol-3-phosphate dehydrogenase (GPDH), translation elongation factor-1 alpha (TEF), beta-tubulin (TUB), calmodulin (CMD), actin (ACT). Isolations yielded 204 fungal isolates belonging to nine different genera, with most isolates belonging to Alternaria and Fusarium. Molecular identification revealed that the isolates belong to 20 fungal species, the most dominant of which was Alternaria alternata. Pathogenicity tests were conducted on each plant species. The inoculations on P. vulgaris revealed that Pythium aphanidermatum induced rotting, damping-off and wilt symptoms while Fusarium equiseti induced yellowing symptoms on the leaves. Rhizoctonia solani produced lesions and root rot on Vigna unguiculata while Curvularia muehlenbeckiae and Curvularia caricae-papayae produced root lesions on the roots of V. unguiculata and V. radiata, respectively. Alternaria alternata produced brown symptoms on the tap root of Vicia faba. P. aphanidermatum resulted in a significant reduction in the fresh weight, dry weight and shoot length of Phaseolus vulgaris. The study shows that several fungal species can be found associated with the roots of beans and Vigna unguiculata in Oman and can result in varying disease symptoms. This is the first report of root lesions produced by Curvularia muehlenbeckiae on the roots of V. unguiculata and by C. caricae-papayae on V. radiata worldwide

Assessment of aflatoxin B1 content and aflatoxigenic molds in imported food commodities in Muscat, Oman

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus are considered as serious food safety and human health issues due to their hepatotoxic effects. In the present study, the occurrence of aflatoxin B1 (AFB1), the most potent human liver carcinogen, and prevalence of toxigenic isolates of Aspergillus spp. were assessed in 140 food commodities in Muscat markets, Oman, and the 95 quarantined imported food commodities. These samples consisted of rice, corn, peanut, red chilli powder, soybean, dried dates and tree nuts. AFB1 was analyzed using competitive ELISA/LC-MS and the aflatoxigenic fungi were detected using plating technique followed by molecular identification. No AFB1 was detected in 89 (63.6%) samples collected from local markets, while 44 (31.4%) samples contained 1-5 ppb and the remaining 7 (5%) samples (red chili powder) contained 6-10 ppb. None of the samples exceeded the maximum permissible limit of 10 ppb set for foods by Oman legislation. Of the 95 quarantined samples, only 17 (17.9%) samples were positive and contained AFB1 at concentrations ranging from 1-3.4 ppb. Four isolates of Aspergillus pp. were isolated from the collected samples and were identified as Aspergillus flavus (A14, A16 and A23) and A. chevalieri (A46) on the basis of internal transcribed spacer (ITS) sequences of ribosomal DNA. Among them, A. flavus strain A14 alone produced AFB1 (7.6 ppb), while A16, A23, and A46 were non-toxigenic. This is the first detailed report on the occurrence of AFB1 in food commodities imported into Oman

Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus

Wet bubble disease (WBD) caused by Hypomyces perniciosus is a major constraint of button mushroom (Agaricus bisporus) cultivated worldwide. A few synthetic chemical fungicides are used to control WBD. In our study, the potential of essential oil (EO) from Zataria multiflora in inhibition of H. perniciosus was evaluated as an alternative to chemical fungicides. An isolate of H. perniciosus was isolated from wet bubble diseased A. bisporus and pathogenicity of the mycoparasite was determined under artificially inoculated conditions. The mycoparasitic fungus was identified using sequences of the internal transcribed spacer (ITS) region of ribosomal DNA. The EO was extracted from the aerial parts of Z. multiflora by microwave extraction method and evaluated in vitro for its antifungal activity against H. perniciosus. The EO of Z. multiflora (ZEO) at the tested concentrations (50% and 100%) inhibited the growth of H. perniciosus in the agar diffusion test. The minimum inhibitory concentration (MIC) of ZEO was 0.04% as assessed by the poisoned food technique. The chemical composition of ZEO was determined by gas chromatography-mass spectrometry analysis. A total of 23 compounds were identified. Among them, the most abundant compounds were Linalool (20.3%) and Bornyl acetate (15.5%). Linalool at the tested concentrations of 0.25% and 0.125% completely inhibited the mycelial growth of H. perniciosus in an in vitro assay. These results suggest that ZEO can be exploited for control of WBD

The effect of salt-tolerant antagonistic bacteria from tomato rhizosphere on plant growth promotion and damping-off disease suppression under salt-stress conditions

A total of 24 morphologically distinct salt-tolerant bacteria were isolated from the tomato rhizosphere soil and tested in vitro against Pythium aphanidermatum, causing damping-off disease in tomato. Among them, five bacterial isolates viz., BTR1.0, BTR1.1, BTR4.4, BTR7.0 and BTR8.6 were found to inhibit the mycelial growth of P. aphanidermatum. The maximum antagonistic effect was shown by BTR1.0 followed by BTR7.0 and BTR1.1. Based on the MALDI Biotyper analysis, these bacterial isolates were identified as Serratia marcescens (BTR1.0 and BTR1.1), Pseudomonas aeruginosa (BTR7.0 and BTR8.6) and Alcaligenes faecalis (BTR4.4). Analysis of plant growth stimulating activity of bacterial strains revealed that A. faecalis BTR4.4 recorded the highest vigour index followed by S. marcescens BTR1.0. The biocontrol activity of bacterial antagonists against tomato damping-off was tested under salt-water irrigation. Among the bacterial strains tested, S. marcescens BTR1.1 was found to be highly effective under non-saline water irrigation, which suppressed the disease by 68% compared to infected control. However, at 50 mM NaCl concentration, S. marcescens strain BTR1.0, P. aeruginosa strain BTR7.0 and A. faecalis BTR4.4 provided a significant level of control of the disease. At 100 mM NaCl concentration, no significant effect of the bacterial strains on the disease incidence was observed

Genetic analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom on acid lime trees

“Candidatus Phytoplasma aurantifolia” is associated with witches’ broom disease of lime in Oman and the UAE. A previous study showed that an infection by phytoplasma may not necessarily result in the physical appearance of witches’ broom symptoms in some locations in Oman and the UAE. This study investigated whether phytoplasma strains belonging to “Ca. P. aurantifolia” (based on the 16S rRNA gene analysis) in locations where disease symptoms are expressed are different from phytoplasma in locations where disease symptoms are not expressed. About 21 phytoplasma strains (15 from areas and trees with disease symptoms and six from areas and trees without disease symptoms) were included in the analysis. The study utilized sequences of the imp and SAP11 genes to characterize the 21 strains. Phylogenetic analysis of both genes showed that the 21 strains are similar to each other and to reference strains in GenBank. The study shows that there is a low level of diversity among all phytoplasma strains. In addition, it shows that phytoplasma in places where witches’ broom symptoms are not expressed are similar to phytoplasma in places where disease symptoms are expressed. This may suggest that disease expression is not linked to the presence of different phytoplasma strains, but may be due to other factors such as weather conditions

Potential of indigenous antagonistic rhizobacteria in the biological control of Monosporascus root rot and vine decline disease of muskmelon

Monosporascus root rot and vine decline (MRVD) of muskmelon, caused by Monosporascus cannonballus, is an economically important disease worldwide. The objectives of this study were to isolate native rhizobacterial strains and to evaluate their ability to promote plant growth and to provide protection against M. cannonballus. Thirty eight native bacterial isolates from the rhizosphere soil of muskmelon and cucumber were screened for their antagonism against M. cannonballus in a dual culture assay. Among them, five isolates viz., Bacillus amyloliquefaciens (B4), Pseudomonas mendocina (B7), Bacillus endophyticus (B10), Pseudomonas resinovorans B11 and P. aeruginosa AT3, identified based on the 16S rRNA gene sequence analysis, showed a significant level of antagonism and the inhibition zone ranged from 5.6 mm to 25.9 mm. Scanning electron microscopic observation of the hyphae of M. cannonballus at the inhibition zone revealed morphological abnormalities including shrinkage, loss of turgidity, pit formation and deformation. These bacterial isolates showed compatibility with one another and with Trichoderma viride. Greenhouse experiments revealed that P. resinovorans B11 was the most effective among the bacterial antagonists in controlling MRVD in melon. When applied as seed treatment and soil application, P. resinovorans B11 reduced the incidence of MRVD by 93.1%, relative to the infected control

Occurrence, characterization and management of fruit rot of immature cucumber fruits under arid greenhouse conditions

A study was undertaken to characterize and manage pathogens associated with fruit rot of immature cucumber fruits in greenhouses in Oman. A survey over 5 growing seasons from 2008 to 2010 in 99 different greenhouses in Oman showed that the disease is prevalent in 91 (92%) greenhouses and results in losses of 10 to 60% (avg. 33%) of immature fruits per plant. Incidence of the disease was not found to be affected by growing seasons, which could be attributed to the limited fluctuations in ambient temperatures in greenhouses. Isolations from diseased cucumber fruits yielded Alternaria alternata (isolation frequency = 52%), Fusarium equiseti (40%), Cladosporium tenuissium (27%), Botrytis cinerea (6%), Fusarium solani (6%), Corynespora cassiicola (3%), Aspergillus spp. (2%), Curvularia sp. (1%) and Bipolaris sp. (1%). With the exception of Curvularia and Bipolaris species, all other fungi were pathogenic on cucumber fruits, with Fusarium equiseti being the most aggressive, followed by Corynespora cassiicola, Botrytis cinerea and Alternaria alternata. Cladosporium and Aspergillus spp. were found to be weakly pathogenic. Comparing the efficacy of foliar and soil applications of carbendazim fungicide on fruit rot of cucumber showed that foliar applications significantly reduced fruit rot and increased cucumber yield when compared to soil application or to control (P < 0.01). This appears to be the first report of the association of Corynespora cassiicola and Fusarium equiseti with fruit rot of immature greenhouse cucumbers. This is also the first report in Oman for the association of Cladosporium tenuissimum with fruit rot of immature cucumbers. Findings are discussed in terms of factors affecting disease control in greenhouses using carbendazim

In vitro antagonistic potential, plant growth-promoting activity and indole-3-acetic acid producing trait of bacterial isolates from spent mushroom substrate of Agaricus bisporus

Spent mushroom substrate (SMS) is widely used as fertilizer and to control plant diseases. The microorganisms surviving in SMS play a crucial role in plant growth promotion and biocontrol properties of SMS. In this study, an effort was made to isolate and characterize the bacterial species present in the SMS of Agaricus bisporus and to study their antagonistic potential, plant growth-promoting ability and indole-3-acetic acid (IAA) producing trait. Six different bacterial isolates exhibiting morphological variabilities were obtained from the SMS by serial dilution technique. On the basis of 16S rRNA gene sequences, these isolates were identified as Staphylococcus epidermidis (Sh1 and Sh3), S. aureus (Sh2), Bacillus albus (Sh4), Delftia lacustris (Sh6) and Comamonas aquatica (Sh7). These bacterial strains were assayed for their antagonism against Pythium aphanidermatum, a phytopathogenic oomycete. The results of in vitro dual culture assay revealed that all the 6 bacterial isolates showed low levels of suppression of P. aphanidermatum and recorded less than 5 mm inhibition zone. Among the bacterial isolates, S. epidermidis Sh3 recorded the maximum inhibition zone of 4.2 mm. Plant growth promotion test using roll paper towel method revealed that C. aquatica Sh7, B. albus Sh4, D. lacustris Sh6 and S. epidermidis Sh3 caused a significant increase in seedling vigour of cucumber compared to control. The seeds treated with the bacterial isolate C. aquatica Sh7 showed the maximum seedling vigor. Assessment of in vitro production of IAA by the bacterial isolates revealed that the bacterial isolates highly varied (ranging from 0.28 to 9.25 mg L-1) in their potential for production of IAA. The maximum amount of IAA was produced by C. aquatica Sh7 (9.25 mg L-1), while the minimum was produced by S. epidermidis Sh1 (0.28 mg L-1)

In vitro detoxification of aflatoxin B1 by aqueous extracts of medicinal herbs

Aflatoxin contamination in food commodities is a major food safety concern all over the world. These aflatoxins are a group of carcinogenic secondary metabolites produced by some fungi belonging to Aspergillus section Flavi. To minimize the level of aflatoxins in food commodities, a wide range of pre- and post-harvest procedures have been employed. In this study, aqueous extracts of 13 medicinal herbs were assessed for their ability to detoxify aflatoxin B1 (AFB1), the most potent and commonly occurring carcinogen in foods. Among them, herbal extracts of Hybanthus enneaspermus, Eclipta prostrata and Centella asiatica exhibited over 70% detoxification of AFB1. The degradation of AFB1 upon treatment with these herbal extracts was confirmed by liquid chromatography–mass spectrometry analysis. Two fractions (Rf 0.75 and 0.87) purified from H. enneaspermus by using thin-layer chromatography displayed in vitro AFB1-detoxifying properties. Gas chromatography–mass spectrometry analysis of the active fractions revealed the presence of linalool and bornyl acetate as the major components suggesting possible involvement of volatile compounds of H. enneaspermus in the detoxification of AFB1. To our knowledge, this is the first report on the detoxification of AFB1 by H. enneaspermus, E. prostrata and C. asiatica aqueous extracts