First Report of Neofusicoccum australe Causing Branch Dieback of English Walnut cv. Chandler in Central Chile.

The English walnut (Juglans regia L.) is the second most important fruit crop of importance in Chile, with 43,700 hectares mainly in the Central Valley (www.odepa.cl, 2022). For several seasons symptoms of a branch dieback have been observed in walnut orchards with 3 to 50% of trees incidence levels. During the 2020 winter season (July) a total of 150 symptomatic spurs of 15 trees were sampled from an 8-year-old walnut cv. Chandler orchard located in Buin (33°42' S, 70° 42' W). The collected spurs showed external and internal brown necroses, starting from the tip with well-defined margins. The symptomatic tissue was cut in to small pieces (5 x 4 x 2 mm), surface disinfected by dipping in a 10% solution made from a commercial bleach solution (4,9% NaOHCl), rinsed twice in sterile water and plated on APDA (PDA Difco laboratories acidified with lactic acid (2,5 ml of 25% (vol/vol) per liter of medium). After five days at 20 °C in darkness, fast-growing, white-grey turning to black colonies were obtained, tentatively classified as a member of the Botryosphaeraceae family and two single-spore isolates (SS1, SS2) were selected for identification. Colony mycelia were first white and turned to light grey, dark grey or black, with tufts of mouse gray aerial mycelia. The pycnidia and conidia production was induced by inoculating autoclaved pine needles placed on APDA an incubation for 25 to 30 days at 20 °C in darkness. Black pycnidia solitary and globose were obtained producing hyaline, aseptate, fusiform to obovoid conidia with truncated ends with dimensions of (22.6-) 19.1 ± 1.4 (-13.3) x (6.7-) 5.5 ± 0.5 (-3.7) µm and 3.5 length/width ratio (n=100). Both isolates were identified using dichotomous keys confirming the description of Crous et al, 2006 as Neofusicoccum australe. The identification was molecularly confirmed by amplifying the nuclear ribosomal gene 5,8S (ITS1-5.8S-ITS2) using the ITS1/ITS4 primers, a partial region of ß-tubulin gene (Bt2a/Bt2b), and the translation elongation factor 1-α gene (TEF1) with TEF1-728F/TEF1-986R primers. The BLASTn search revealed 100% of identity for ITS and TEF according to sequences of N. australe reference strains MT587467.1 and MK759852.1, respectively; and over 99% for ß-tubulin compared to N. australe strain KX464929.1. The DNA sequences were submitted to the GenBank (ITS, OP142414, OP142416; BT, OP209981, OP209978; and TEF OP209979, OP209980) for SS1 and SS2 isolates, respectively, and deposited in the fungal collection of CChRGM - INIA, Chillán, Chile (RGM 3409 and 3410). Pathogenicity of both isolates was tested in 8-year-old asymtomatic English walnut cv. Chandler in the field during 2020 spring season, by cutting transversally 15 twigs of different tress and inoculating with a 5 day-old PDA plug. An equal number of wounded twigs were inoculated with a sterile PDA plug and served as control. After six months, all inoculated twigs developed the same necrotic lesions observed in field of 2.0 to 10.1 cm (SS1) and 1.9 to 10.8 cm (SS2) in length while control twigs showed only a scar without any dieback tissues. The inoculated pathogens of N. australe were recovered from the diseased tissues, thus fulfilling Koch's postulates. A similar dieback of walnut was reported in Chile, which caused Diplodia mutila (Díaz et al, 2018), and N. parvum (Luna et al, 2022) while N. australe has been reported in other hosts (Auger et al, 2013, Besoain et al, 2013). To the best of our knowledge, this is the first report of N. australe associated with walnut branch dieback in Chile.

Potential biological control of the pupal stage of the European grapevine moth Lobesia botrana by the entomopathogenic fungus Beauveria pseudobassiana in the winter season in Chile.

OBJECTIVE: Lobesia botrana, the European grapevine moth, affects Vitis vinifera L. and other species of economic importance in a number of countries through damage caused by its larvae in berries and associated secondary diseases such as Botrytis cinerea. Control of the moth in urban areas is difficult due to poor chemical management of infested plants in houses. Additionally, in winter, L. botrana is in its pupal stage covered with a cocoon that prevents the penetration of chemical pesticides. For this reason, the objective of this work was to control the pupal stage with a formulation based on the entomopathogenic fungus Beauveria pseudobassiana in urban areas. RESULTS: The strain RGM 1747 was identified as B. pseudobassiana by multilocus sequence analysis. The biocontrol activity of this formulated fungus against the infestation of vines with breeding pupae without cocoons showed 100% infection 21 days after inoculation under winter conditions. Finally, the biocontrol activity of the formulated fungus against natural infestations of L. botrana in winter in urban areas reached an efficacy of 51%. This result suggests that the B. pseudobassiana formulation is able to penetrate the cocoon and contributes to the integrated pest management of L. botrana.

Expression of an optimized Argopecten purpuratus antimicrobial peptide in E. coli and evaluation of the purified recombinant protein by in vitro challenges against important plant fungi.

Antimicrobial peptides (AMP) have been widely described in several organisms from different kingdoms. We recently designed and evaluated a synthetic version of an AMP isolated and characterized from Argopecten purpuratus hemocytes. This study describes the generation of a chimaeric gene encoding for Ap-S, the use of this construct to transform E. coli strain BL21, and the evaluation of the purified recombinant Ap-S (rApS) as an antifungal agent. The proposed gene coding for rAp-S consists of 93 nucleotides arranged downstream from the IPTG-inducible T7 promoter. The best synthesis conditions were obtained after E. coli cultivation at 26°C for 3h, which allowed for the production of an rAp-S-enriched fraction containing the peptide at 249µM. Mass spectrometry analysis of the purified rAp-S (3085.80Da) showed the addition of a glycine residue on its N-terminal end derived from vector design and peptide purification. The purified rApS fraction was assayed for antifungal activity by direct addition of purified rApS elution to potato dextrose agar media at a final concentration of 81nM. These assays showed important growth inhibitions of both biotrophic (Fusarium oxysporum, Trichoderma harzianum) and necrotrophic (Botrytis cinerea, Alternaria spp.) fungi in that the hyphae structures and spore count were affected in all cases. The strategy of cloning and expressing rAp-S in E. coli, the high yield obtained and its successful use for controlling pathogenic fungi suggest that this molecule could be applied to agricultural crops using various management strategies.