Assessment of Inoculation Methods of Thielaviopsis paradoxa (De Seynes) Höhn into Oil Palm Seedlings under Greenhouse Conditions

Oil palm (Elaeis guineensis Jacq. and Elaeis Oleifera Cortes) is one of the most important oil crops in the world. Colombia is the fourth-largest oil palm producer worldwide. However, oil palm diseases are a significant factor affecting yield. Thielaviopsis paradoxa (De Seynes) Höhn is a pathogen that affects young palm trees, causing spear rot. Four disease establishment methods were studied to replicate, in a controlled environment, the symptoms of the disease found in the field. Young palm trees were inoculated with a suspension of endoconidia using either local infiltration, drip, scissor cut, or direct contact with agar blocks bearing mycelia and conidia. The effects of the inoculation methods were studied in dose-method-disease severity experiments conducted in a greenhouse under controlled conditions. All four methods resulted in T. paradoxa infections and the development of symptoms of the disease. The disease severity was correlated with the method and dose of inoculation. In trials to test Koch’s postulates, T. paradoxa was isolated from areas of disease progression in the inoculated trees, but the teleomorph Ceratocystis paradoxa (Dade) Moreau was not observed. A photographic record of the infection process at different times post-infection was compiled. Given that establishing the disease through artificial inoculation is essential for assessing plant pathogenesis, this study determined that the local infiltration method (1 × 106 endoconidia mL−1) and a 3–7 day incubation period were critical for the development of symptoms as severe as those observed in natural infections in the field.

When toxic chemicals refuse to die—An examination of the prolonged mercury pesticide use in Australia

Mercury, even in low concentrations, is known to cause severe adverse human health effects. In the early 1900s, mercury became a popular fungicide ingredient, leading to multiple poisoning incidents that forced much of the world to act upon phasing out mercury use in agriculture. These incidents spurred the advancement of mercury science and the implementation of international policies and regulations to control mercury pollution worldwide. Despite these developments internationally, Australia continued using methoxyethyl mercury chloride as a fungicide to treat sugarcane against the fungi Ceratocystis paradoxa (pineapple disease). At the request of the manufacturer and following pressure from Australian researchers and the Minamata Convention on Mercury, Australian authorities announced a ban on mercury-containing pesticide in May 2020. Australia’s unique reluctance to act on controlling this hazardous pollutant makes it an interesting case study for policy inaction that runs counter to global policy trends and evidence-based decision making. As such, it can provide insights into the challenges of achieving multilateral agreement on difficult environmental issues such as global warming. In this review, I discuss the scientific development and policy decisions related to mercury fates and exposure of wildlife and humans in Australia to mercury used in pesticide. The historical uses of mercury pesticide and poisoning incidents worldwide are described to contextualize Australia’s delayed action on banning and controlling this chemical product compared to other nations. Regulations on mercury use in Australia, which has not ratified the Minamata Convention on mercury, are compared to those of major sugarcane and pesticide producer nations (Brazil, China, Japan, India, Thailand, and United States) which have ratified the Convention and replaced mercury pesticides with alternative products. I discuss how mercury regulations have the potential to protect the environment, decrease human exposure to mercury, and safeguard the ban on mercury products. Ratifying the Minamata Convention would give Australia equal footing with its international counterparts in global efforts to control global mercury pollution.

Genetic Transformation and Green Fluorescent Protein Labeling in Ceratocystis paradoxa from Coconut

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a ‘reporter’ gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.

Diagrammatic scale of severity for postharvest black rot (Ceratocystis paradoxa) in coconut palm fruits

ABSTRACT One of the major bottlenecks in the postharvest commercialization of coconut fruits is the black rot disease, caused by the fungus Ceratocystis paradoxa. This disease has been neglected in the main production areas or associated with other coconut diseases. To date, there are no standardized methods for assessing the intensity of such a problem. In this sense, the aim of our study was to prepare and validate a diagrammatic scale to evaluate the disease severity in coconut fruits. We also tested the scale applicability comparing distinct C. paradoxa isolates on inoculated fruits. For such quantification, the fruit longitudinal sectioning was necessary, allowing the visualization of internal symptoms. The proposed scale showed good repeatability and high reproducibility, with absolute errors around 8%, while the coefficient of determination presented an average of 91% with the aid of the scale and of 59% without the scale. In addition, symptom analyses showed that the outer necrosis does not match the internal damage. Therefore, we suggest in this study that the severity estimate of this disease be obtained by using the presented scale.

Antifungal Activity of the Extract and the Active Substances of Endophytic Nigrospora sp. from the Traditional Chinese Medicinal Plant Stephania kwangsiensis

To exploit a new source from medical plants for finding bioactive products, endophytic fungi DBR-5 identified as Nigrospora sp., was isolated from the root tubers of the traditional Chinese medicinal plant Stephania kwangsiensis Lo. The antifungal activities of the extract from its fermentation liquids were determined. The ethyl acetate extract of DBR-5 exhibited high and broad antifungal activities against plant pathogenic fungi, and showed high toxicity to Exserohilum turcicum, Bipolaris maydis, Ceratocystis paradoxa, Alternaria oleracea and Cochliobolus miyabeanus with EC50 values respectively at 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.03 mg/mL and 0.04 mg/mL. By a bioassay guided fractionation, three antifungal secondary metabolites were isolated from liquid culture of DBR-5, and identified as griseofulvin, deoxybostrycin and austrocortirubin on the basis of spectroscopic analysis. In vitro antifungal assay showed that griseofulvin displayed significant inhibition against the hypha growth of tested plant pathogenic fungi with EC50 values ranging from 0.0013 mg/mL to 0.0202 mg/mL, and showed the highest toxicity to E. turcicum and C. paradoxa with EC50 values both at 0.0013 mg/mL. Compared with the broad spectrum fungicide carbendazim, except that the toxicity of griseofulvin to Diaporthe citri and Pestalotiopsis theae was lower, the toxicity to the other eight pathogenic fungi was much higher. The inhibitory rates of griseofulvin against spore germination of A. olerace, C. paradoxa and P. theae were 100%, 100% and 94.39% respectively, at a concentration of 0.01 mg/mL. The other two compounds deoxybostrycin and austrocortirubin exhibited only weak antifungal activities. The results indicate the potential of Nigrospora sp. DBR-5 as a source of griseofulvin and also support that griseofulvin is a natural compound with high potential bioactivity against plant pathogenic fungi.

ANTIFUNGAL APPRAISAL OF BURKHOLDERIA GLADIOLI STRAIN VIMP03 (JQ867372) AGAINST CERATOCYSTIS PARADOXA

ABSTRACTObjective: The purpose of the present investigation was to assess antifungal characterization of Burkholderia gladioli strain VIMP03 (JQ867372), anisolate from sugar beet rhizosphere.Methods: Antifungal characterization was carried out by biochemical, dual culture, and agar well diffusion methods against Ceratocystis paradoxa, asoilborne pathogen of sugarcane and other crops. Culture filtrate and ethyl acetate extract obtained from culture supernatant were analyzed by highperformanceliquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses, respectively.Results: The culture filtrate and ethyl acetate extract exhibited effective antifungal activity. Organic acid profile of the culture filtrate was determined.Acetic acid was mainly produced by the culture under study. The GC-MS analysis of ethyl acetate extract documented the presence of compoundsincluding tetratetracontane, 9-nonadecene, erucic acid, and other hydrocarbon derivatives.Conclusion: The GC-MS, HPLC, and biochemical profiles of B. gladioli strain VIMP03 (JQ867372) revealed its agro clinical-antifungal potential.Keywords: Antifungal, Burkholderia gladioli, High-performance liquid chromatography, Gas chromatography-mass spectrometry.