Evaluation of the biocontrol potential of an antagonist against olive knot disease in olive trees (Olea europaea L.)

The olive knot, caused by the bacterium Pseudomonas savastanoi pv. savastanoi (Psv), is one of the most serious diseases affecting olive tree (Olea europaea L.) in many regions of the world and mainly in Mediterranean countries. This disease produces tumors, especially in olive tree stems and trunk, leading to the death of the plant. In recent years, the use of microbial biocontrol agents for the control of plant diseases has gained considerable attention. Previous studies have revealed that, several bacteria strains naturally occurring in the olive tree phyllosphere, exhibit capacity to inhibit in vitro growth of Psv, being the greatest antagonistic effect displayed by Bacillus En40. The aim of the present study was to evaluate the ability of this strain to suppress Psv and olive knot development in olive trees. Pot experiments were conducted with 2-year-old olive plantlets cv. Cobrançosa inoculated with the selected strain or Psv, or in combination of both. Phenotypic traits associated with plant growth (e.g. biomass yield, total shoot/root height, and photosynthetic pigments content) and biocontrol abilities (e.g. abundance of Psv and assessment of disease symptoms) were evaluated, up to 120 days after inoculation. While development and growth of Psv+antagonist-inoculated plants showed to be similar to non-inoculated plants, photosynthetic pigments contents were observed to be higher in antagonist-inoculated plants than in other treatments. When compared with Psv-inoculated plants, plants treated with the antagonist revealed a significant reduction (p<0.01) in knots formation and size. Antagonist-inoculated plants showed also a reduction of the abundance of Psv in the inoculation sites (from 22.4 to 7.5 UFC/μL, p<0.01), suggesting its capacity to reduce the patogen's growth. This strain was identified to supres Psv and disease development on olive plants and thus, it should be considered in the future as a possible biocontrol agent against olive knot disease.This work is funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012. D. MINA thanks thinfo:eu-repo/semantics/publishedVersio

Biological control of Pseudomonas savastanoi pv. savastanoi by two bacterial isolated from olive tree phyllosphere

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi (Psv), has been responsible for high economical crop losses in olive orchards, especially in Mediterranean countries. Olive knot cannot be eradicated once it is established in an orchard, and therefore its control is based on preventive measures. Laboratory experiments previously performed by us have showed the capacity of some bacterial species, isolated from olive tree phyllosphere, to inhibited Psv growth. In the present work, the two most promising bacterial isolates (i.e. Frondihabitans sp. and Paenibacillus sp.) were evaluated for the control of Psv in olive plantlets (Olea europaea L.) under greenhouse conditions, to predict their effect in a more natural condition. In pot experiments, two-year-old olive plants cv. Cobrançosa were inoculated with the antagonistic bacteria and Psv individually or in combination. Inoculations were performed in wounds previously made in three different sites of the main stem. Thirty replicate plants were used per strain. The plants were observed for symptom development and the number of bacteria on the inoculation sites was periodically evaluated, for up to 120 days after inoculation. To quantify the reduction of symptom expression, knots were excised from stems and their weights were compared between treatments. Inoculation with Psv resulted in the formation of knots with significantly higher weight values compared to plants inoculated simultaneously with Psv and antagonistic bacteria. Both tested bacterial also showed the capacity to reduce the amount of Psv in the inoculation sites, suggesting their effectiveness in controlling the multiplication of the pathogen. Data presented here demonstrate for the first time the potential of these bacteria for suppression of olive knot, and these two species should be considered in the future as potential biocontrol agents against Psv.This work is funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012. D. MINA thanks the Fundação para a Ciência e Tecnologia (FCT), Portugal for the Ph.D. grant SFRH/BD/105341/2014info:eu-repo/semantics/publishedVersio

Endophytic and epiphytic phyllosphere fungal communities are shaped by different environmental factors in a Mediterranean ecosystem

The diversity and potential factors that influence fungal assemblages in the phyllosphere ofMediterranean tree species have been barely studied, especially when endophytic and epiphytic communities are simultaneously considered. The endophytic and epiphytic fungal communities in the phyllosphere of olive tree, a typically Mediterranean species adapted to grow under adverse climatic conditions, were studied for evaluating differences between both fungal communities, and examine whether different abiotic (climate-related) and biotic (plant organ) factors play a role in structuring these communities. Both communities differed in size and composition, being the epiphytic community richer and more abundant, displaying a dominance of melanized fungi. Season was the major driver of community composition, ~ especially of epiphytes. Other drivers shaping epiphytes were wind speed and temperature. Plant organ, rainfall, and temperature were drivers of endophytic composition. In contrast, limited variation is provided by canopy orientation, but its effect was distinct among seasons. In conclusion, epiphytic and endophytic communities are not driven by the same factors. Several sources of variation interact in complex ways to form and maintain the phyllosphere fungal community in Mediterranean climates. The importance of climatic parameters for these fungal communities suggests that they are likely to be affected by future climate change.info:eu-repo/semantics/publishedVersio

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

Susceptibility of Cydia splendana to the entomopathogenic fungus Beauveria bassiana

Os fungos entomopatogénicos têm-se revelado uma alternativa promissora aos produtos químicos usados na luta contra pragas. Apesar do grande esforço empreendido na sua utilização, o seu efeito num grande número de pragas é ainda desconhecida. O bichado da castanha, Cydia splendana L., é a principal praga do castanheiro no norte de Portugal, causando prejuízos consideráveis. A utilização de meios de luta químicos apresenta limitações devido, por um lado, ao número reduzido de substâncias homologadas, e por outro porque a utilização de pesticidas de síntese não se coaduna com o modo de produção sustentável em que os soutos são conduzidos na região. Adicionalmente os meios de luta culturais e biotécnicos têm-se revelado de difícil implementação e de eficácia reduzida. Com o presente trabalho pretendeu-se avaliar a susceptibilidade de C. splendana ao fungo entomopatogénico Beauveria bassiana. Testou-se o efeito de quatro isolados (A39GF09; A67GF09; LC39GF10 e PA95GF10) da colecção do IPB, obtidos de larvas do lepidóptero Prays oleae recolhidos na região de Trás-os- Montes. Para tal em laboratório, foram inoculadas larvas de C. splendana, pelo método de superfície, com seis concentrações de conídios (1 x 105; 1 x 106; 5 x 106; 1 x 107; 5 x 107; 1 x 108). Os quatro isolados de B. bassiana testados demonstraram capacidade para infectar as larvas de C. splendana. A mortalidade confirmada apresentou uma correlação linear positiva com a concentração de conídios, embora com diferenças entre isolados. O Tempo Letal médio (LT50) e a Concentração Letal média (LD50) foram também variáveis entre isolados. A LD50 variou entre < 1 x 105 (LC39GF10) e 2,13 x 105 (A67GF09), enquanto que LT50 variou entre < 5 e 10 dias. A acção dos isolados de B. bassiana testados sobre C. splendana, apesar da grande variabilidade observada, abre a possibilidade do seu uso na luta biológica contra esta praga do castanheiro.Entomopathogenic fungi are one of the most promising alternatives to the use of chemical pesticides. Although a considerable amount of knowledge is available on this kind of fungi, their effect in a great number of pests is still unknown. The chestnut fruit moth, Cydia splendana L., is one of the major pests of chestnut in northern Portugal, able to cause severe damage. The chemical control has several limitations either due to the limited number of approved substances or because the use of chemical pesticides is not consistent with the sustainable mode of production in which the groves are conducted in the region. Additionally, both cultural and biotechnical controls have proven to be difficult to implement and to have reduced efficacy. This works intends to evaluate the susceptibility of C. splendana to the entomopathogenic fungus Beauveria bassiana. The four isolates tested (A39GF09; A67GF09; LC39GF10 and PA95GF10) were obtained from the IPB collection and were isolated from Prays oleae larvae collected in the Trás-os-Montes region. For this, C. splendana larvae were inoculated, following the surface method, with six concentrations of conidia (1 x 105; 1 x 106; 5 x 106; 1 x 107; 5 x 107; 1 x 108). Larvae of C. splendana showed to be susceptible to infection by all four B. bassiana isolates. A concentration-dependent mortality was observed, although with variation between isolates. Median lethal time (LT50) and median lethal concentration (LD50) also varied between isolates. LD50 varied from < 1 x 105 (LC39GF10) to 2.13 x 105 (A67GF09) while LT50 ranged from < 5 to 10 days These results show an expected variation between isolates of B. bassiana, and, more importantly, the susceptibility of C. splendana to this entomopathogenic fungus, which open up the possibility of its used to control this particular pest.Ivo Oliveira agradece à Fundação para a Ciência e Tecnologia (FCT), POPH-QREN e FSE pela bolsa de doutoramento concedida (SFRH/BD/44265/2008). Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PTCD/AGRAAM/ 102600/2008.info:eu-repo/semantics/publishedVersio

Illuminating Olea europaea L. endophyte fungal community

A wide array of fungal endophytes is known to inhabit plant tissues and were recently recognized as essential for plant health. A better description of the scarcely known endophyte microbiota in olive tree phyllosphere is the first step for elucidating the microbial interactions that lead to olive disease establishment. In this work, the fungal endophytic community of the phyllosphere of different olive tree cultivars (Cobrançosa, Galega vulgar, Madural, Picual, Verdeal Transmontana) is revealed by using a metabarcoding strategy targeting ITS1 barcode. A total of 460 OTUs were obtained, increasing the broad view of fungal endophytes inhabiting the olive tree phyllosphere, in particular yeast endophytes. New endophytes were persistently found in all cultivar tissues. Different olive tree cultivars depicted distinct endophyte communities. Olive cultivars exhibited dissimilar amounts of fungi with distinct ecological functions, which could explain at least in part their differential susceptibility/tolerance to olive diseases.This work was supported by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundaç˜ao para a Ciˆencia e a Tecnologia) in the framework of the projects EXCL/AGR-PRO/0591/2012, PTDC/AGR-PRO/4354/2012, and PTDC/ASP-PLA/31133/2017, as well as BioISI (UIDB/04046/2020) and Mountain Research Center - CIMO (UIDB/00690/2020).info:eu-repo/semantics/publishedVersio

The effect of olive knot disease in bacterial communities associated to olive phyllosphere

The interactions between microorganisms sharing the same phyllosphere environment can be highly relevant for plant health. Although such interactions are ubiquitous in nature, the possible interaction between the incoming pathogen and the resident microbial flora, as well as the impact of these interactions on disease development/suppression, are unknown and not understood. To assess this, we will use in this study the olive knot disease as a model system. This disease is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (Psv), that produced knots, mainly on olive tree twigs and young branches. Endophytic and epiphytic bacterial were isolated from asymptomatic twigs and knots of olive trees from two cultivars with different susceptibilities to olive knot (cv. Verdeal- Transmontana - susceptible; cv. Cobrançosa - tolerant). Bacterial isolates obtained were molecularly identified through sequencing of V1 - V4 regions from 16S rDNA. Overall, 73 species belonging to 37 genera were identified. The composition of bacterial community colonizing asymptomatic twigs changed drastically when becomes diseased. A set of bacterial was showed to be highly associated with either asymptomatic twigs or knot of each cultivar, suggesting that they might h also to be extremely different between cultivars, suggesting an effect of the cultivar in bacterial community assemblage. Future research should be carried out in order to identify it these bacterial-bacterial and bacterial-plant interactions can be really part of host defense against olive knot disease.This work is funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds by FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012. D. MINA thanks the Fundação para a Ciência e Tecnologia (FCT), Portugal for the Ph.D. grant SFRH/BD/105341/2014info:eu-repo/semantics/publishedVersio

Biological potential of fungi associated to Prays oleae in Trás-os-Montes (Northeastern region of Portugal)

Olive groves represent an important fraction of the agricultural practices in Portugal, mainly in the region of Trás-os-Montes. Our work pretends to disclose the fungal diversity associated to one of the major olives pests, the Prays oleae Bern. Besides evaluating the extent of the presence of entomopathogenic fungi, we also intend to reveal other promising fungi with potential to be used as biological control agents. In order to achieve this goal, larvae and pupae of the three generations of P.oleae were collected from several orchards. Whenever a fungal agent was associated to the cause of death, we proceeded to the preparation of fungal pure cultures, DNA isolation, amplification and sequencing of their ITS region for species identification. High fungal diversity was found, with the majority of the fungi presenting simultaneously entomopathogenic, antagonistic or phytopathogenic characteristics. 22 different species were identified, being Beauveria bassiana, a well-known entomopathogen, the most frequently isolated fungi. The ones described as antagonistic were tested in dual cultures for antagonistic activity against phytopathogenic fungi

Fungal diversity associated to Prays oleae in Trás-os-Montes (Northeastern region of Portugal). A survey of potential entomopathogenic fungi

Olive groves are one of the main agricultural activities in the Portuguese region of Trás-os-Montes. They occupy a very large area, where new biological agricultural practices have been increasingly applied. In order to control the pests that attack this culture, using methods that do not rely on chemical substances, an effort must be conducted to achieve new processes. One of the most important pest in the olive groves in this region is the olive moth (Prays oleae Bern.), responsible for high losses in olive yields. One of the promising approaches to control this pest is the use of entomopathogenic fungi that naturally occur in the olive orchards and are able to infect and kill olive moths. The present work intends to evaluate the diversity of fungal species associated to P. oleae in several olive orchards located in Trás-os-Montes region. To achieve this goal, we collected larvae and pupae of the three annual generations (phyllophagous, antophagus and carpophagus) of P. oleae. Whenever a fungal agent was associated to the cause of death of the moth, we proceeded to the in vitro isolation of the fungal specimen. Pure cultures of each fungus were obtained and were molecularly identified by sequencing the amplified internal transcribed spacer (ITS) region of rDNA. The higher diversity of fungal species was found in the phyllophagous generation, followed by the carpophagus and antophagus. In the phyllophagous generation, 77% of the total isolates were entomopathogenic fungi, whereas 18% were phytopathogenic fungi, being the remaining described as antagonistic fungi. Among the entomopathogenic fungi, the most frequent isolated species (97%) was the mitosporic ascomycete Beauvera bassiana. In the antophagus generation, 55% of the total isolates were phytopathogenic fungi, 41% were antagonistic fungi and only 4% corresponded to entomopathogenic fungus. In the carpophagus generation, more than 50% of the total isolates were phytopathogenic fungi. In this work, it will be discussed the extent of the fungal presence in P. oleae, as well as the characteristics of each fungal species and their applicability in pest contro