Insights into Asparaginase from Endophytic Fungus Lasiodiplodia theobromae: Purification, Characterization and Antileukemic Activity

Endobiotic fungi are considered as a reservoir of numerous active metabolites. Asparaginase is used as an antileukemic drug specially to treat acute lymphoblastic leukaemia. The presented study aims to optimize the media conditions, purify, characterize, and test the antileukemic activity of the asparaginase induced from Lasiodiplodia theobromae. The culture medium was optimized using an experiment designed by The Taguchi model with an activity ranging from 10 to 175 IU/mL. Asparaginase was induced with an activity of 315 IU/mL. Asparaginase was purified with a specific activity of 468.03 U/mg and total activity of 84.4 IU/mL. The purified asparaginase showed an approximate size of 70 kDa. The purified asparaginase showed an optimum temperature of 37 °C and an optimum pH of 6. SDS reduced the activity of asparaginase to 0.65 U/mL while the used ionic surfactants enhanced the enzyme activity up to 151.92 IU/mL. The purified asparaginase showed a Km of 9.37 µM and Vmax of 127.00 µM/mL/min. The purified asparaginase showed an IC50 of 35.2 ± 0.7 IU/mL with leukemic M-NFS-60 cell lines and CC50 of 79.4 ± 1.9 IU/mL with the normal WI-38 cell line. The presented study suggests the use of endophytic fungi as a sustainable source for metabolites such as asparaginase, provides an opportunity to develop a facile, eco-friendly, cost-effective, and rapid synthesis of antileukemic drugs, which have the potential to be used as alternative and reliable sources for potent anticancer agents.

Systemic Identification and Functional Characterization of Common in Fungal Extracellular Membrane Proteins in Lasiodiplodia theobromae

Plant pathogenic fungi deploy secreted proteins into apoplastic space or intracellular lumen to promote successful infections during plant-pathogen interactions. In the present study, fourteen CFEM domain-containing proteins were systemically identified in Lasiodiplodia theobromae and eight of them were functionally characterized. All eight proteins were confirmed to be secreted into extracellular space by a yeast signal peptide trapping system. The transcriptional levels of most CFEM genes, except for LtCFEM2 and LtCFEM6, were significantly elevated during infection. In addition, almost all LtCFEM genes, apart from LtCFEM2, LtCFEM3, and LtCFEM6, were transcriptionally up-regulated at 35°C in contrast to that at 25°C and 30°C. As two elicitors, LtCFEM1 induced local yellowish phenotype and LtCFEM4 triggered cell death in Nicotiana benthamiana leaves. Furthermore, these proteins displayed distinct subcellular localizations when expressed transiently in N. benthamiana. Moreover, two genes, LtCFEM7 and LtCFEM8, were found to be spliced alternatively by RT-PCR and sequencing. Therefore, our data suggest that LtCFEM proteins play important roles in multiple aspects, including pathogenicity and plant immune response, which will enhance our understanding of the sophisticated pathogenic mechanisms of plant opportunistic pathogen L. theobromae.

Response of different grapevine cultivars to infection by Lasiodiplodia theobromae and Lasiodiplodia mediterranea

Botryosphaeria dieback is a grapevine trunk disease that affects all viticulture regions of the world. Species of the genus Lasiodiplodia have been reported as pathogenic towards grapevine in several growing regions and have also been previously reported from Portuguese vineyards. Species in this genus, particularly Lasiodiplodia theobromae, have been reported on previous studies to be more aggressive than other Botryosphaeriaceae species most commonly associated with Botryosphaeria dieback. The aim of this study was to assess the response of some of the more representative cultivars planted throughout Portuguese vineyards, Touriga Nacional, Touriga Franca, Alvarinho, Aragonez (=Tempranillo) and Cabernet Sauvignon, by performing artificial inoculations with Lasiodiplodia spp. collected in different geographic locations worldwide. Two experiments, one by inoculating two-year-old grapevines kept on a greenhouse-controlled conditions with six isolates of L. theobromae and one isolate of L. mediterranea and other by inoculating seven-year-old field grown grapevines with two isolates of L. theobromae, were conducted twice. Response of the cultivars was assessed by evaluating the lesion length caused by the isolates under study, five months after inoculation. The results showed that all isolates studied were able to infect the annual shoots since they were always re-isolated and produced internal wood discoloration. Significant differences were found for all isolate/cultivar combinations. For both experiments, Touriga Nacional showed the largest lesions while Aragonez recorded the smallest lesions amongst the whole lot of cultivars inoculated with Lasiodiplodia spp.. In general, Portuguese isolates were more aggressive than those from Peru, which demonstrated to be mildly aggressive. These results give a first insight on the response of selected Portuguese cultivars to Lasiodiplodia species, which are present in Portugal, but not commonly associated with Botryosphaeria dieback. This contributes to improve knowledge of the impact that Botryosphaeria dieback causal agents have on crucial national cultivars, which may help winegrowers not only to manage current cultural practices, but also to optimize decision making when planning the establishment of new vineyards.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

Cultural Practices Triggering the Development of Citrus Stem Rot in District of Bangli

Kabupaten Bangli, Bali merupakan salah satu sentra produksi jeruk di Indonesia. Kendala pada produksi jeruk di antaranya ialah penyakit busuk pangkal batang yang disebabkan oleh Lasiodiplodia theobromae. Tujuan penelitian ini menentukan faktor budi daya, letak geografis, dan curah hujan yang berkaitan dengan penyakit ini. Pengamatan kondisi penyakit dan wawancara mengenai teknik budi daya ditanyakan kepada 50 petani pemilik. Keterkaitan antara teknik budi daya dan keparahan penyakit dianalisa dari data korespondensi yang dipetakan dengan grafik biplot dan analisis kontingensi dengan uji khi kuadrat. Berdasarkan pada analisis korespondensi dan kontingensi terdapat 4 faktor cara budi daya dan 1 faktor tanaman yang berkaitan nyata terhadap keparahan busuk batang jeruk. Faktor tersebut ialah frekuensi penyiangan secara mekanis, umur tanaman, populasi tanaman per hektar, aplikasi herbisida, dan dosis pupuk nitrogen.

Antifungal activity of crude extracts of Ageratum conyzoides and Chromolaena odorata for management of Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae through in vitro evaluation

Lasiodiplodia is an important genus of fungi causing destructive diseases on perennial crops, including cocoa. Two crucial species of Lasiodiplodia that cause diseases in cocoa are Lasiodiplodia theobromae and Lasiodiplodia pseutheobromae. A variety of weeds is the potential to be applied as botanical fungicides to control the pathogens. The main objective of this study was to evaluate Ageratum conyzoides and Chromolaena odorata leaf extract to inhibit the growth of L. theobromae and L. pseudotheobromae on a synthetic medium. Solvent organic was methanol for weed extraction with a ratio of 1:5. The experiment was conducted through the poison food technique method, both in the solid and liquid medium in three different concentrations, 1, 3, and 5%. The result showed that A. conyzoides and C. odorata were significantly inhibited the colony growth of both Lasiodiplodia in all concentrations in a solid medium. A. conyzoides performed better than C. odorata in all concentrations of both Lasiodiplodia in inhibition. A. conyzoides 5% performed well to suppress the colony growth of L. pseudotheobromae (100%), followed by A. conyzoides 3% and A. conyzoides 1%. A. conyzoides 5% able to inhibit the colony growth of L. theobromae until 100%, followed by A. conyzoides 3% and 1%. Meanwhile, A. conyzoides and C. odorata extract tested on PDB medium at 1, 3, and 5% reduced the fungal biomass significantly at all concentrations. C. odorata was found most effective in inhibiting fungal biomass of both pathogens either on wet weight or on dry weight at 1, 3, and 5% %. A. conyzoides and C. odorata can manage the growth of L. theobromae and L. pseudotheobromae through in vitro conditions.

A Novel Lipase from Lasiodiplodia theobromae Efficiently Hydrolyses C8-C10 Methyl Esters for the Preparation of Medium-Chain Triglycerides’ Precursors

Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer’s disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8–C10 methyl esters (C8–C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8–C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8–C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8–C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1’s well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs.