Biocontrol of postharvest pathogens infecting avocado using endophytic trichoderma species.

Abstract

Pre-harvest fungal infection causes both pre- and postharvest avocado (Persea americana Mill.) diseases. Some foliar diseases also cause avocado fruit spots, including those caused by Pseudocercospora and Cladosporium species. Pre- and postharvest rots of avocado fruit are largely caused byColletotrichum, Lasiodiplodia and Neofusicoccum species, and these are also latent pathogens that usually infect the fruit in the orchards before harvest. These fungal pathogens cause crop losses in SouthAfrican production in untreated fruit by about 50-90%. As a result, several agrochemicals including prochloraz, copper oxychloride, thiophanate-methyl and thiabendazole are being used by farmers for avocado disease management. However, the intensive use of chemicals does not prevent all diseases, and toxic residues affect the environment and consumers of fruit containing residues. As a result, there are growing restrictions on the use of agrochemicals being exported to the European Union (EU), due to reductions in theMaximum Residue Levels (MRLs) of most of these fungicides. The use of biological control agents (BCAs) such as Trichoderma species has been regarded as a promising and environmentally friendly approach to controlling plant diseases. Trichoderma species are opportunistic, avirulent plant symbionts and some strains have been developed commercially as BCAs for use on many crops against a wide range of plant pathogens. Their modes of action are complex and include antibiosis, mycoparasitism, producing bioactive secondary metabolites, inducing plant defensive mechanisms and promoting plant growth. Therefore, the overall objective of this study was to isolate and screen endophytic strains of Trichoderma species to control fruit diseases of avocado such as stemend rot, anthracnose and leaf spot diseases. Isolation and Identification of Fungal Pathogens Avocado fruit from different supermarkets in Pietermaritzburg (KwaZulu-Natal, South Africa) that displayed symptoms of avocado fruit rot and fruit spot were collected and brought to the Plant Pathology Laboratory at the University of KwaZulu-Natal for fungal isolation. A total of forty-five isolates were isolated from symptomatic avocado fruit with typical characteristics of anthracnose, stem-end rot, Cladosporium spot, and Cercospora spot. The isolates were identified based on their cultural and morphological characteristics using light microscopy and scanning electron microscopy (SEM). Koch’s postulates screening was performed on fresh avocado fruit by spraying the fruit with suspensions of the key fungal pathogens of anthracnose, stem-end rot, and leaf spot. Frequently isolated colonies causing anthracnose on avocado fruit were divided into 2 morphological groups (Cs1 and Cs2). Colonies of Group 3 (Ls1) constituted 35% of the fungal pathogens isolated from fruit displaying stemend rot. Group 4 (Ls2) was a Lasiodiplodia species that was provided by Majola (2020) and freshly isolated colonies of this genus. In Group 5 were isolates of Pseudocercospora and Cladosporium species, which constituted about 20% of the cultures.

All the fungal isolates were subjected to a pathogenicity assay, conducted twice, to confirm the pathogenicity of each isolate. Anthracnose pathogens in Group 1 (Cs1) were the most virulent strains, while Group 2 (Cs2) were the least virulent. Stem-end rot pathogens in Group 3 (Ls1) were more virulent compared to Group 4 strains (Ls2). Some inoculated fruit did not show any symptoms, even after 14 days post inoculation (dpi). Morphological characterization showed that Pseudocercospora species and Cladosporium spp. were the least virulent strains. Molecular identification of the fungi was undertaken using ITS sequence analysis. Isolate Cs1 was identified as Colletotrichum cobbittiense and isolate Cs2 was identified as Colletotrichum henanense. The most virulent isolate causing stem-end rot was Isolate Ls1, which was identified as Neofusicoccum parvum, a pathogen that was not previously recognized as being of importance in KwaZulu-Natal, South Africa. This suggested that this fungus is probably causing severe postharvest losses for avocado farmers in KwaZulu-Natal. Isolate Ls2 was identified in a previous study as Lasiodiplodia mahajangana using ITS1 and ITS2 gene sequence analysis. Isolation and Endophytic Screening of Trichoderma species Avocado leaves were sampled from the five avocado trees of the cultivar Fuerte, which are growing at Ukulinga farm, University of KwaZulu-Natal, Pietermaritzburg. From these trees, sixty leaves were sampled, and five fresh avocado fruits from a local supermarket in Scottsville, Pietermaritzburg were used as plant material for the isolation of Trichoderma species. Colony colour differences and radial growth measurements were two characteristics that were used to differentiate between the strains of Trichoderma species. A screening for endophytic activity of the isolated Trichoderma strains was conducted on fresh avocado fruit and seedlings. Fifty-two avocado seedlings of the cultivar Edranol were transplanted into pots in a greenhouse and sprayed with benomyl to kill any natural endophytic Trichoderma spp. found in them. The seventeen Trichoderma strains isolated previously were used to prepare suspensions with a concentration of 1x106 conidia/ml-1. The Trichoderma suspensions were sprayed on the seedlings and after two weeks the seedling leaves were sampled randomly and surfacesterilised using 2% sodium hypochlorite and sterile distilled water. Small segments were placed on Trichoderma selective medium (TSM) agar plates to check for the presence of the Trichoderma. During this study, only eleven strains of Trichoderma spp. were found to be

endophytic. Another screening was then done on fresh avocado fruits. The fruits were sprayed with suspensions of the eleven endophytic Trichoderma strains. After a seven-day waiting period, the fruit was surface-sterilized, and the skin of the fruit was sampled in ten places per fruit and plated onto TSM. Pure cultures were purified on Potato Dextrose Agar (PDA) and monitored every second day to record the growth. A total of nine isolates of Trichoderma demonstrated high endophytic ability. Assessment of the Effect of Endophytic Trichoderma Strains on Anthracnose and Stem-end rot in vivo An in vivo investigation was conducted to understand the antagonistic activity of endophytic Trichoderma as a potential biocontrol agent against anthracnose and stem-end rot. Fresh avocado fruit of the cultivar Hass were collected from a local farm in Richmond (KwaZulu-Natal, South Africa). The fruit were sprayed with Trichoderma isolates conidial suspensions at concentration 1x106 conidia/ml-1 and a commercial Trichoderma-based product (Eco77). The fruit were then air-dried and stored in boxes inside black plastic bags to create a high relative humidity at room temperature for 7 days. After 7 days post inoculation (dpi), the black plastic bags were removed and the fruit were inoculated with the pathogens by spraying the isolates Cs1, Cs2, Ls1, and Ls2 with concentration 1x106 conidia/ml-1. This experiment was conducted twice to confirm the antagonistic activity of the endophytic Trichoderma isolates. A rating scale of 1-3 was used to describe the disease severity caused by isolates Cs1, Cs2, Ls1, and Ls2 after the fruit were treated with endophytic Trichoderma strains. All the fruit inoculated with Trichoderma and the pathogen developed less anthracnose or stem-end rot symptoms than fruit inoculated with the pathogen only. Isolates UK1E, UK4C and Eco77 were able to reduce symptoms on the fruit inoculated with the pathogenic species isolated, including C. cobbittiense, C. henanense, N. parvum and L. mahajangana. Based on ITS1 and ITS2 gene sequence analysis, UK1E was identified as Trichoderma asperellum and UK4C as Trichoderma koningiopsis. These two strains have the potential to be commercialized as biocontrol agents against the Botryosphaeriaceae family associated with anthracnose and stem-end rot of avocado. The next research phase would be to undertake field trials to see if preventative inoculation of the best Trichoderma strains can provide seasonal protection of fruit from pre- and postharvest pathogens.