Evaluation of indigenous fungal entomopathogens and aqueous leaf extract of Annona muricata against Polyphagotarsonemus latus infesting Jatropha curcas in Indonesia

Puspitarini RD, Afandhi A, Fernando I. 2021. Evaluation of indigenous fungal entomopathogens and aqueous leaf extract of Annona muricata against Polyphagotarsonemus latus infesting Jatropha curcas in Indonesia. Biodiversitas 22: 2648-2655. The broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) has been known to cause a great deal of injury on Jatropha curcas (L.) plantation in Indonesia. Isolation of indigenous fungal entomopathogens from rhizosphere soils, followed by pathogenicity assay was conducted to find an effective isolate for controlling P. latus. Additionally, the potential combination of the selected isolate and soursop (Annona muricata L.) aqueous leaf extract (SLE) was investigated. A total of 24 isolates were obtained by using the insect bait method. Four fungal isolates, namely Paecilomyces sp. 1., Lecanicillium sp., Beauveria sp., and Fusarium sp. 1, showed high conidial viability and were chosen as representatives to assess their pathogenicity against P. latus. Among the tested isolates, Beauveria sp. which had the highest conidial viability among the tested isolates, needed a shorter period to completely kill the tested mites. However, the compatibility test revealed the deleterious effect of SLE on Beauveria sp. SLE at all concentrations showed a very toxic effect on the fungus, therefore the two must be applied separately. Our results provide useful information on the effectiveness of indigenous entomopathogenic fungi Beauveria sp. and aqueous leaf extract of A. muricata as an alternative tool to control the broad mite P. latus on the J. curcas plantation.

First report of Alternaria alternata causing leaf spot on Oat (Avena sativa) in India

Oat (Avena sativa L.) is an important cereal crop grown worldwide primarily for food and animal feed. In November 2019, a leaf spot disease was observed on the oat plants at Mandya (12.5218° N, 76.8951° E), Karnataka, India. The disease incidence on plants was ranged between 43 to 57 percent. Initially, the symptoms appeared on leaves as small dark-brown spots surrounded by a yellow halo later turned to irregular necrotic spots with a yellow halo. A total of thirty leaves showing typical symptoms were collected from ten plants (three leaves per plant), cut into an area of 4-5 mm pieces at the junction of infected and healthy tissues. Cut tissues were soaked in 75% ethanol for 45 seconds, followed by 1% sodium hypochlorite solution for 1 min, rinsed five times in sterile distilled water, air dried, and placed on PDA and incubated at 25 ± 1 ℃. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for five isolates. The colonies initially produced light-greyish aerial mycelia, then turned to dark-greyish upon maturity. Conidia were obclavate to pyriform and measured 17.34 to 46.97 µm long, 5.38 to 14.31 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2.73 to 10.17µm) (n = 50.) Based on the morpho-cultural characteristics, the isolates were identified as Alternaria spp., and PCR assay using species-specific primers (AAF2/AAR3; Konstantinova, et al. 2002) confirmed the taxonomic identity of all five isolates as A. alternata. To further confirm the identity, the internal transcribed spacer (ITS), small subunit (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), an anonymous gene region OPA10-2, KOG1058 and translation elongation factor 1-alpha (tef1) of two isolates MAAS-1 and MAAS-2 were PCR amplified using the primers described previously (Woudenberg et al. 2015; Praveen et al. 2020) and the resultant PCR products were sequenced and deposited in NCBI GenBank (ITS: MW487388, MW741962, SSU: MW506220, MW752854, gapdh: MW506221, MW752855, rpb2: MW506222, MW752856, Alt a1: MW506223, MW752857, endoPG: MW506224, MW752858, OPA10-2: MW506225, MW752859, KOG1058: MW506226, MW752860, and tef1: MW506227, MW752861) which showed (99.62%, 99.81%), (100%, 100%), (100%, 99.66%), (100%, 100%), (99.58%, 99.15%), (99.55%, 99.32%), (99.53%, 99.68%), (99.23%, 99.56%) and (99.17%, 99.58%) identity with ITS (AF347031), SSU (KC584507), gapdh (AY278808), rpb2 (KC584375), Alt a1 (AY563301), endoPG (JQ811978), OPA10-2 (KP124632), KOG1058 (KP125233) and tef1 (KC584634) genes/genomic regions of type strain CBS916.96 of A. alternata respectively, confirming the identity of MAAS-1 as A. alternata. For pathogenicity assay, the conidial suspension (2 × 106 conidia/ml) of MAAS-1 isolate was artificially sprayed until runoff on ten healthy oat plants (cv. Kent, 35 days old) and ten plants sprayed with sterile water served as control. All plants were covered with polyethylene covers and kept under the greenhouse at 28 ± 1 ℃. The pathogenicity assay was repeated three times. After six days post-inoculation, small dark-brown spots with light-yellow halo appeared on leaves of MAAS-1inoculated plants. In comparison, no symptoms were observed on control plants. The fungal pathogen was re-isolated from the artificially infected plants and confirmed as A. alternata based on morpho-cultural characteristics and PCR assays. The leaf spot disease of Oat caused by A. alternata has already been reported from China (Chen et al. 2020); to our knowledge, it is the first report of A. alternata causing leaf spot on Oat in India. The leaf spot disease is an emerging threat to oat cultivation, and it reduces the grain yield and leaf quality; therefore, its management is essential for increasing productivity.

Complete genome sequencing of Pseudomonas syringae pv. actinidiae Biovar 3, P155, kiwifruit pathogen originating from China

Pseudomonas syringae pv. actinidiae is a bacterial pathogen of kiwifruit. Based on the results of the pathogenicity assay, we sequenced the strain Pseudomonas syringae (Psa3) P155 which possesses a series of virulence and resistance genes, CRISPR candidate elements, prophage related sequences, methylation modifications, genomic islands as well as one plasmid. Most importantly, the copper resistance genes copA, copB, copC, copD, and copZ as well as aminoglycoside resistance gene ksgA were identified in strain P155, which would pose a threat to kiwifruit production. The complete sequence we reported here will provide valuable information for a better understanding of the genetic structure and pathogenic characteristics of the genome of P155.

A Rapid Pathogenicity Assay of R. solanacearum in 10-12 Days Old Eggplant (Solanum melongena) Seedlings by Root Inoculation Approach

1.AbstractThis study has reported standardization of pathogenicity assay of Ralstonia solancearum in eggplant seedlings by root inoculation approach. Though the approach is similar to my earlier published work that done with tomato seedlings, in case of eggplant, duration of the seed germination, age of the seedlings, symptom timing and progression rate of the disease are different. In an introductory observation, R. solanacearum F1C1 inoculation was performed in to 10-12 days old eggplant seedlings resulted in lethal infection in eggplant seedlings and subsequently most of these infected seedlings got wilted within 10 days of post-inoculation. Colonization studies of R. solanacearum in wilted as well as infected eggplant seedlings were confirmed by GUS staining as well as fluorescence microscopy. In addition, we also observed that wrapping of the eggplant seedlings root by a thin layer of cotton soon after the pathogen inoculation in the root, enhanced the disease progression and wilting of the inoculated seedlings. The standardized root inoculation protocol in eggplant seedlings was found to be efficient to distinguish mutant strains of different virulence genes such as hrpB, phcA and pilT from wild-type R. solanacearum. Due to its reproducibility and consistency in different eggplant cultivars, the standardized protocol described here is practically observed as an useful and rapid approach to investigate R. solanacearum pathogenicity and disease progression at the early seedling stage of eggplant.

Soybean Sudden Death Syndrome Causal Agent Fusarium brasiliense Present in Michigan

Sudden death syndrome (SDS), caused by members of Fusarium solani species complex (FSSC) clade 2, is a major and economically important disease in soybean worldwide. The primary causal agent of SDS isolated to date in North America has been F. virguliforme. In 2014 and 2016, SDS symptoms were found in two soybean fields located on the same farm in Michigan. Seventy Fusarium strains were isolated from roots of the SDS-symptomatic soybeans in two fields. Phylogenetic analysis of partial sequences of elongation factor-1α, the nuclear ribosomal DNA intergenic spacer region, and the RNA polymerase II beta subunit revealed that the primary FSSC species isolated was F. brasiliense (58 and 36% in each field) and the remaining Fusarium strains were identified as F. cuneirostrum, F. phaseoli, an undescribed Fusarium sp. from FSSC clade 2, and strains in FSSC clade 5 and FSSC clade 11. Molecular identification was supported with morphological analysis and a pathogenicity assay. The soybean seedling pathogenicity assay indicated that F. brasiliense was capable of causing typical foliar SDS symptoms. Both root rot and foliar disease severity were variable by strain, just as they are in F. virguliforme. Both FSSC 5 and FSSC 11 strains were also capable of causing root rot, but SDS foliar symptoms were not detected. To our knowledge, this is the first report of F. brasiliense causing SDS in soybean in the United States and the first report of F. cuneirostrum, F. phaseoli, an as-yet-unnamed Fusarium sp., and strains in FSSC clade 5 and FSSC clade 11 associated with or causing root rot of soybean in Michigan.

investigation of fungi infesting the caraway seeds (Carum carvi L.) in the south region of Poland

Studies on the incidence the caraway seeds for pathogen infestation and test the pathogenicity of isolated fungi to caraway seedlings were carried out. A total of 383 fungi were isolated from seeds with evident symptoms of disease and without symptoms of disease. In this case a complex of different pathogens was isolated, among which Alternaria, Fusarium and Epicoccum were predominant. Pathogenicity assay on caraway seedlings was performed using isolates originating from caraway seeds. All tested fungi showed a very high aggressiveness in pathogenicity test.