Morphological, pathogenic and molecular characterization of Lasiodiplodia theobromae: a causal pathogen of black rot disease on kenaf seeds in Malaysia

Kenaf (Hibiscus cannabinus) is a fibre crop grown in Malaysia as a substitute crop for tobacco. Previous study have recorded that kenaf has been infected by various genera of seed-borne pathogen include Fusarium, Synnematium, Alternaria, Colletotrichum and Botrytis. Seed-borne disease affects and actively attacks seeds and may be harmful. Lasiodiplodia theobromae is a seed-borne fungal pathogen that infects a variety of crop seeds. Studies on the isolation of seed-borne fungi on kenaf seed have revealed that L. theobromae causes black rot disease on kenaf seeds. L. theobromae was successfully isolated from kenaf seeds on an agar plate and a blotter. L. theobromae was isolated frequently from infected seeds and identified based on its cultural and morphological characteristics. The fungus sequence was analysed using molecular technique (ITS-rDNA amplification). A pathogenicity test was used to confirm that L. theobromae caused blackening of the seeds and reduced the germination against a control treatment in potato Dextrose Agar (PDA) medium. To our knowledge, this study is the first to confirm that L. theobromae is the causal agent of black rot on kenaf seed in Malaysia

Screening for active compounds in Rhus coriaria L. crude extract that inhibit the growth of Pseudomonas syringae and Ralstonia solanacearum

An experiment was performed to study the antibacterial activity of methanol, acetone, alcohol and aqueous extracts from the fruit of R. coriaria by disk diffusion assay in terms of minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and killing-time curve. The detection of the components was also fulfilled using Gas Chromatography–Mass Spectrometry (GC-MS) and also tested for their antibacterial activity. The tested bacteria were Pseudomonas syringae (Accession No. KJ858057), a tomato bacterial speck causal agent, and Ralstonia solanacearum (Accession No. KJ881159) causing tomato bacterial wilt. Furthermore, the inhibition criteria were made by different extracts of the sampled bacteria which were measured and compared with standard antibiotic (chloramphenicol). Aqueous extract displayed better outcomes against P. syringae and R. solanacearum as compared to chloramphenicol. According to the GC-MS test results, the aqueous extract was composed of 39 different phytocompounds, together with eight elements in the high peak region, namely Furfural, 1-Cyclopetene, 2,5 Furandione, Phloroglucinol, Succinic acid, Malic acid, P-Tolylacetic acid and Coumalic acid. Among these, it was discovered that 2,5 Furandione was the most important antibacterial element that is present in sumac. The results from the current study indicate that different extracts of R. coriaria contain a variety of antibacterial compounds which can potentially be used to produce an extensive range of herbal mixtures with anti-bacterial properties for controlling diseases in crops belonging to the Solanaceae family

First report of leaf blight on Parthenium hysterophorus caused by Nigrospora sphaerica in Malaysia

Weeds may act as inoculum reservoirs for fungal pathogens that could affect other economically important crops (Karimi et al. 2019). In February 2019, leaves of the ubiquitous invasive weed, Parthenium hysterophorus L. (parthenium weed) exhibiting symptom of blight were observed at Ladang Infoternak Sg. Siput (U), a state-owned livestock center in Perak, Malaysia. Symptoms appeared as irregularly shaped, brown-to-black necrotic lesions across the entire leaf visible from both surfaces, and frequently on the older leaves. The disease incidence was approximately 30% of 1,000 plants. Twenty symptomatic parthenium weed leaves were collected from several infested livestock feeding plots for pathogen isolation. The infected tissues were sectioned and surface-sterilized with 70% ethyl alcohol for 1 min, rinsed three times with sterile distilled water, transferred onto potato dextrose agar, and incubated at 25°C under continuous dark for 7 days. Microscopic observation revealed fungal colonies with similar characteristics. Mycelium was initially white and gradually changed to pale orange on the back of the plate but later turned black as sporulation began. Conidia were spherical or sub-spherical, single-celled, smooth-walled, 12 to 21 μm diameter (mean = 15.56 ± 0.42 μm, n= 30) and were borne on a hyaline vesicle. Based on morphological features, the fungus was preliminarily identified as Nigrospora sphaerica (Sacc) E. W. Mason (Wang et al. 2017). To confirm identity, molecular identification was conducted using isolate 1SS which was selected as a representative isolate from the 20 isolates obtained. Genomic DNA was extracted from mycelia using a SDS-based extraction method (Xia et al. 2019). Amplification of the rDNA internal transcribed spacer (ITS) region was conducted with universal primer ITS1/ITS4 (White et al. 1990; Úrbez-Torres et al. 2008). The amplicon served as a template for Sanger sequencing conducted at a commercial service provider (Apical Scientific, Malaysia). The generated sequence trace data was analyzed with BioEdit v7.2. From BLASTn analysis, the ITS sequence (GenBank accession number. MN339998) had at least 99% nucleotide identity to that of N. sphaerica (GenBank accession number. MK108917). Pathogenicity was confirmed by spraying the leaf surfaces of 12 healthy parthenium weed plants (2-months-old) with a conidial suspension (106 conidia per ml) collected from a 7 day-old culture. Another 12 plants served as a control treatment and received only sterile distilled water. Inoculation was done 2 h before sunset and the inoculated plants were covered with plastic bags for 24 h to promote conidial germination. All plants were maintained in a glasshouse (24 to 35°C) for the development of the disease. After 7 days, typical leaf blight symptoms developed on the inoculated plants consistent with the symptoms observed in the field. The pathogen was re-isolated from the diseased leaves and morphological identification revealed the same characteristics as the original isolate with 100% re-isolation frequency, thus, fulfilling Koch's postulates. All leaves of the control plants remained symptomless and the experiment was repeated twice. In Malaysia, the incidence of N. sphaerica as a plant pathogen has been recorded on several important crops such as watermelon and dragon fruit (Kee et al. 2019; Ismail and Abd Razak 2021). To our knowledge, this is the first report of leaf blight on P. hysterophorus caused by N. sphaerica from this country. This report justifies the significant potential of P. hysterophorus as an alternative weed host for the distribution of N. sphaerica

Spatial modelling of papaya dieback disease occurrence

The incident of papaya dieback disease has resulted in huge losses due to the decrement in crop yield. The disease is affecting papaya production in Malaysia, putting papaya production at risk, primarily to accommodate the national and export trading needs. Abiotic factors have been identified as agents influencing the occurrence of papaya disease, however, little study includes the possible influence of landscape features on disease occurrence. Therefore, using a spatial model, this study aims to investigate the influence of weather variables and the surrounding landscape on papaya dieback disease in Batu Pahat district, Johor, Malaysia. This study applied an Ordinary Least Square (OLS) regression to identify the dominant abiotic factors influencing the papaya disease incident. The main finding revealed that the distance of disease incidents significantly influences the rate of papaya dieback disease based on the affected points (crop) to the nearby road; the percentage area of papaya dieback disease incidence was negatively related to the distance from the road. In other words, being closer to road features may increase the size of the affected area. This research could benefit stakeholders in strategising agricultural practices including planning suitable landscape and topographic characteristics of newly planted areas to reduce the occurrence of such disease

Effect of temperature on germination, radial growth, and sporulation of the new isolates of Metarhizium anisopliae and their virulence to Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Metarhizium anisopliae is a potential entomopathogenic fungi (EPF) to control insect pests in Malaysia, yet little is known about the temperature influence on its growth and efficacy. The average daily temperature in Malaysia is between 21 °C and 32 °C, with some parts of Cameron Highland in Peninsular Malaysia experiencing temperatures as low as 14 °C. Therefore, for effective field application, the most effective EPF isolates should be tolerant to the temperature of the country. Here we conducted a laboratory experiment to determine the effects of average daily temperatures in Malaysia (15, 20, 25, and 30 °C) on conidial germination, radial growth, and conidial density of seven new isolates of M. anisopliae. However, at 25 and 30°C, the three isolates (Ma-2, Ma-9, and Ma-15) produced the highest number of conidia and were therefore selected for virulence test against the second instar of whitefly, Bemisia tabaci. The percentage mortality of the three isolates ranged between 36.1% and 97.7% across different temperatures for 7 days post-treatment. The isolates Ma-15 caused the highest percentage mortality of 97.7% and the shortest LT50 and LT90 values (1.68 and 3.98 days, respectively), at 30 °C incubation temperature. This study confirms that the growth and virulence of Malaysian isolates of M. anisopliae are strongly influenced by temperature and could be used as promising candidates for biocontrol of B. tabaci on vegetables planted in a tropical climate such as that of low land and highland in Malaysia, although extensive field and semi-field bioassays, as well as the development of ideal formulation, are still required

Effect of temperature on germination, radial growth, and sporulation of the new isolates of Metarhizium anisopliae and their virulence to whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Metarhizium anisopliae is a potential entomopathogenic fungi (EPF) to control insect pests in Malaysia, yet little is known about the temperature influence on its growth and efficacy. The average daily temperature in Malaysia is between 21 °C and 32 °C, with some parts of Cameron Highland in Peninsular Malaysia experiencing temperatures as low as 14 °C. Therefore, for effective field application, the most effective EPF isolates should be tolerant to the temperature of the country. Here we conducted a laboratory experiment to determine the effects of average daily temperatures in Malaysia (15, 20, 25, and 30 °C) on conidial germination, radial growth, and conidial density of seven new isolates of M. anisopliae. However, at 25 and 30°C, the three isolates (Ma-2, Ma-9, and Ma-15) produced the highest number of conidia and were therefore selected for virulence test against the second instar of whitefly, Bemisia tabaci. The percentage mortality of the three isolates ranged between 36.1% and 97.7% across different temperatures for 7 days post-treatment. The isolates Ma-15 caused the highest percentage mortality of 97.7% and the shortest LT50 and LT90 values (1.68 and 3.98 days, respectively), at 30 °C incubation temperature. This study confirms that the growth and virulence of Malaysian isolates of M. anisopliae are strongly influenced by temperature and could be used as promising candidates for biocontrol of B. tabaci on vegetables planted in a tropical climate such as that of low land and highland in Malaysia, although extensive field and semi-field bioassays, as well as the development of ideal formulation, are still required

Selecting antagonistic yeast for postharvest biocontrol of Colletotrichum gloeosporioides in papaya fruit and possible mechanisms involved

Colletotrichum gloeosporioides causes anthracnose disease in papaya fruit resulting in tremendous economic loss due to its latent infection. This study aimed to evaluate the biocontrol activity of antagonistic yeasts against C. gloeosporioides in papaya and determine the possible mechanism involved. One hundred and ten yeast strains were isolated from different parts of the papaya plant. Among them, only five strains, namely F001, F006, L003, FL013 and LP010, showed more than 55% radial growth inhibition of C. gloeosporioides. These five potent yeast strains were further evaluated in vitro and in vivo. The results indicated that strain F001 had the strongest biocontrol activity based on spore germination and fungal growth inhibition. In Vivo, the strain F001 caused 66.7% and 25% reductions in disease incidence and severity, respectively. Based on molecular identification, the strain F001 was confirmed as Trichosporon asahii. Despite there was no significant induction of defense enzyme activities found on the treated fruits, SEM observation showed direct attachment of T. asahii with the fungal hyphae and interfere in their establishment to the fruit surface. Based on these findings, the antagonistic yeast T. asahii strain F001 may be used as a potential natural biological control agent against anthracnose disease in papaya frui

Genetic diversity of Ralstonia solanacearum phylotype II sequevar 4 strains associated with Moko disease of banana (Musa spp.) in Peninsular Malaysia

Moko disease caused by Ralstonia solanacearum (R. solanacearum) is a major disease affecting banana (Musa spp.) production. Although local reports suggested that this disease is widespread in Malaysia, molecular characterization of R. solanacearum strains associated with Moko disease in this country has not yet been done. During March 2011 to June 2012, 170 banana plants associated with Moko disease and the adjacent soil samples were collected in 12 different locations of five outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan and Johor, with disease incidence exceeding 80 % in some severely affected plantations. A total of 197 strains were identified as R. solanacearum-like colonies since they produced fluidal colonies that were white to pink coloration after incubation at 24 to 48 h at 29 °C on Kelman’s TZC agar medium, appeared as Gram-negative rods, and positive for potassium hydroxide (KOH), Kovacs oxidase, catalase and lipase activity on Tween 80 solution tests. Biovar tests disclosed that only 30 strains displayed characteristics of biovar 1 R. solanacearum associated with Moko disease, which was negative for utilization of disaccharides and hexose alcohols. Pathogenicity assay showed that these 30 strains were virulence towards Musa paradisiaca cv. Nipah explants with diverse degrees of virulence. Phylotype-specific multiplex PCR (Pmx-PCR) revealed all strains belonged to phylotype II displaying a 372 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all 30 strains into phylotype II/4, together with the reference sequences strains from Peru (UW129, UW162 and UW163) and Colombia (UW070). Pooled rep-PCR fingerprinting method defined two major groups; cluster 1 (sub-group A and B) and cluster 2 (sub-group C), with 35 % average similarity coefficient within these two clusters. The sub-groups in cluster 1 were represented by strains from Kedah, Selangor, Negeri Sembilan and Johor; while cluster 2 sub-group was represented exclusively by strains of Pahang. To our knowledge, this is the first description of R. solanacearum phylotype II/4 in Malaysia and the Asian region. Our findings may expand constructive documentation and describe a better understanding on diversity of Malaysian R. solanacearum Moko-causing strains populations, thus will be useful for designing disease control strategies

Actinomycetes as plant growth promoter of rice plants and biocontrol agent against bacterial leaf streak disease

Bacterial leaf streak (BLS) disease caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important rice bacterial diseases. Genotypic and phenotypic similarity of this pathogen to the causal agent of bacterial leaf blight (BLB), Xanthomonas oryzae pv. oryzae (Xoo), has driven an interest on BLS study. Bacterial leaf streak control measures are focusing on the development of resistant varieties and application of copper-based fungicide. Actinomycetes possess a remarkable potential as biological agent. This study was conducted with the following objectives; 1) to characterize the causal pathogen of BLS disease, Xoc and to differentiate it from other Xanthomonas oryzae pathovar, Xoo, 2) to characterize and determine the potential of actinomycetes as biocontrol agent against Xoc and as plant growth promoter and 3) to evaluate the efficacy of actinomycetes treatment on rice growth promotion and BLS suppression through the induction of defense-related enzymes. Five Xoc isolates were discovered in this study and successfully differentiated with Xoo. BLS symptoms were pronounced during early infection stage, yellow small streak lesions can be observed in rice leaves. Xoc can be detected and differentiated with Xoo by multiplex PCR method. Molecular characterization by gyrase subunit B gene amplification had successfully identified all five isolates as Xanthomonas oryzae pv. oryzicola (accession number in GeneBank database (MH560793-MH560797). All isolates developed hypersensitivity reaction on Nicotiana tabacum. Isolate TKC1 is the most virulent isolate, produced 6.78 cm lesion length in pathogenicity assay compared to other four Xoc isolates. Out of 20 Xoc antagonistic actinomycetes, 60% were belonged to various species of Streptomyces. Rhizospheric actinomycetes, SS8 demonstrated the highest (p≤0.05) antagonistic activity with inhibition value of 17.67 mm, followed by TKSC3 (14.00 mm). Isolate SS8 and TKSC3 were identified as Streptomyces sp SW4-2S and Streptomyces shenzhenensis, respectively by 16s rRNA amplification. Both isolates also possess potentials as hydrolytic enzyme producer and plant growth promoter based on in vitro assessment. In vitro assessment with 12 hours seed bacterization with single or consortium treatment of TKSC3 and SS8 isolates revealed significant (p≤0.05) improvement in rice seed germination and seedling vigor performance. TKSC3 and SS8 were both root colonizing and endophytic streptomycetes with average population ranged from 0.66 to 6.52 x 103 CFU/g in seedling roots at 10 days after seed bacterization treatment. Consortium treatment (TKSC3+SS8) exhibited the highest values in plant growth parameters, total chlorophyl, soil and leaf nutrient contents in glasshouse experiment. Consortium treatment (TKSC3+SS8) also demonstrated the highest BLS disease suppression efficiency at 81.02% with the lowest AUDPC value of 95.79. Single and consortium treatments of actinomycetes successfully suppressed BLS disease by enhancing defense-related enzymes accumulation in the rice plant. Peroxidase, polyphenol oxidase, phenyl ammonia lyase and β,1-3 glucanase enzymes activity were increased in actinomycete-treated plants compared to untreated plants which started at 2 days post inoculation. This study confirmed that actinomycetes possess huge potentials as plant growth promoter and biocontrol agent against Xoc pathogen