Exploring common but untapped fungal strains from olive trees to be used as biocontrol agents or plant-growth promoting

The use of plant growth promoting microorganisms, which act directly promoting growth or indirectly as agents of biocontrol of diseases, has been gaining importance as a sustainable tool for increasing agricultural production. In this regard, microorganisms that live in association with plants, either on the surface (epiphytes) or inside (endophytes) their tissues, are seen as the most promising. These beneficial properties have been seen mainly at the level of fungi of the order Phaeomoniellales. Therefore, in this work a collection of fourteen isolates of this order previously obtained from olive leaves was characterized. In particular, we proceeded to their molecular identification using a multi-locus approach and to the evaluation of their biotechnological potential to act as biocontrol agent and plant growth promoting fungi in olive tree. The phylogenetic analysis of six loci, namely internal transcribed spacer region (ITS), actin (ACT), chitin synthase 1 (CHS-1), beta tubulin (β-tubulin), translation elongation factor 1-alpha (TEF-1α) and histone H3 (HIS), allowed to assigned isolates into Phaeomoniellales (eight isolates) and Eurotiales (five isolates) order. Curiously, three of the identified species (Celerioriella umnquma, Pseudophaeomoniella globosa and Pseudophaeomoniella oleae) were reported for the first time in Portugal. The same analysis indicated that five isolates, assigned to Penicillium, were previously misidentified. Penicillium species separation was better when ACT, β-tubulin and/or ITS are simultaneously used; while ITS, TEF-1α and β-tubulin was the best set of loci for resolving Pseudophaeomoniella species. The five Penicillium strains were screened for their ability to produce compounds frequently associated to biocontrol and plant growth promotion traits. Although none of the isolates were able to reduce significantly the in vitro growth of the causal agent of olive anthracnose, Colletotrichum fioriniae, it was found that some isolates increased significantly the production of cellulase, amylase and siderophores, when challenged by the pathogen. All five Penicillium isolates were able to solubilize phosphate and to produce indole acetic acid (IAA). Overall, N172, N162 (Penicillium sp. 2) and N299 (Penicillium sp. 3) isolates were the most promising as biocontrol agents and plant growth promotors. Their effect in in planta assays must be conducted in the future.A utilização de microrganismos promotores do crescimento das plantas, que atuam diretamente promovendo o crescimento ou indiretamente como agentes de biocontrolo, têm vindo a ganhar importância como instrumento sustentável para aumentar a produção agrícola. Neste âmbito, os microrganismos que colonizam as plantas, quer na sua superfície (epífitos) quer no interior dos seus tecidos (endófitos), são vistos como os mais promissores. Estas propriedades benéficas foram observadas principalmente ao nível dos fungos da ordem Phaeomoniellales. Assim, neste trabalho caracterizou-se uma coleção de catorze isolados desta ordem previamente obtidos a partir de folhas de oliveira. Em particular, procedeu-se à sua identificação molecular utilizando uma abordagem multi-locus e à avaliação do seu potencial biotecnológico como agente de biocontrolo e promotor de crescimento na oliveira. A análise filogenética de seis loci, nomeadamente região espaçadora transcrita interna (ITS), actina (ACT), quitina sintase 1 (CHS-1), beta tubulina (β-tubulina), fator de alongamento de tradução 1-alfa (TEF-1α) e histona H3 (HIS), permitiu classificar os isolados na ordem Phaeomoniellales (oito isolados) e Eurotiales (cinco isolados). Curiosamente, três das espécies identificadas (Celerioriella umnquma, Pseudophaeomoniella globosa e Pseudophaeomoniella oleae) foram reportadas, pela primeira vez, em Portugal. A mesma análise indicou que cinco isolados, classificados como sendo Penicillium, foram previamente mal identificados. A separação das espécies de Penicillium foi melhor quando os loci ACT, β-tubulina e/ou ITS foram simultaneamente utilizados; enquanto que a combinação ITS, TEF-1α e β-tubulina foi a melhor para separar as espécies de Pseudophaeomoniella. Os cinco isolados de Penicillium foram estudados quanto à sua capacidade de produzir compostos frequentemente associados ao biocontrolo e à promoção de crescimento das plantas. Apesar de nenhum dos isolados ter mostrado capacidade de reduzir significativamente o crescimento do agente causal da gafa, Colletotrichum fioriniae, em condições in vitro, verificou-se que alguns isolados aumentaram significativamente a produção de celulase, amilase e sideróforos, quando estimulados pelo agente patogénico. Todos os cinco isolados de Penicillium foram capazes de solubilizar fosfato e de produzir ácido indolacético (IAA). No geral, N172, N162 (Penicillium sp. 2) e N299 (Penicillium sp. 3) parecem ser os isolados mais promissores como agentes de biocontrolo e promotores de crescimento das plantas. O seu efeito em ensaios com plantas deve ser testado no futuro

Potential of the endophyte Penicillium commune in the control of olive anthracnose via induction of antifungal volatiles in host plant

Olive anthracnose, caused by several Colletotrichum species, is the most economically harmful fruit disease of the olive crop. This work aimed to evaluate the ability of the endophyte Penicillium commune CIMO 14FM009 to protect the olive tree against Colletotrichum nymphaeae via induction of plant volatile organic compounds (VOCs). Accordingly, olive tree branches were inoculated with the endophyte and one month later with the pathogen. After 0, 3, and 24 h of pathogen inoculation, the volatile composition of leaves and fruits was analyzed by HSSPME- GC/MS, and compared with controls (branches inoculated with buffer, endophyte, or pathogen). The effect of plant-derived volatiles on C. nymphaeae was also evaluated. Penicillium commune induced the release of VOCs on the olive trees, with the capacity to reduce significantly the growth (up to 1.4-fold) and sporulation (up to 1.2-fold) of C. nymphaeae. This effect was most notorious on olives than on leaves, and occurred 3 h after pathogen-challenge, suggesting the need for a stressful stimulus for the production of antifungal VOCs. The observed inhibition was associated to a specific set of VOCs released from olives (mostly belonging to the alcohols and esters chemical classes) and leaves (mostly belonging to the alkenes). Curiously, a set of VOCs belonging to alkene, alkane and ester classes, were emitted exclusively in olive branches inoculated with C. nymphaeae. These findings provide new possibilities for controlling olive anthracnose using P. commune and/or volatiles, which efficacy should be tested in future works.This work is supported by FEDER funds through the COMPETE (Operational Program for Competitiveness Factors) and by National funds through the FCT (Foundation for Science and Technology) in the scope of the project POCI-01–0145-FEDER-031133 “MicOlives - Exploiting plant induced resistance by beneficial fungi as a new sustainable approach to olive crop protection”, Horizon 2020, the European Union’s Framework Programme for Research and Innovation, in the scope of the project PRIMA/0002/2018 “INTOMED- Innovative tools to combat crop pests in the Mediterranean”, and IFAP via the project “Bio4Med - Implementation of innovative strategies to increase sustainability in perennial Mediterranean crops”, as well as the Mountain Research Center - CIMO (UIDB/00690/2020; UIDP/00690/2020) and SusTEC (LA/P/0007/2020).info:eu-repo/semantics/publishedVersio