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

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

The influence of bioclimate on soil microbial communities of cork oak

Background: Soil microbiomes are important to maintain soil processes in forests and confer protection to plants against abiotic and biotic stresses. These microbiomes can be affected by environmental changes. In this work, soil microbial communities from different cork oak Portuguese forests under different edaphoclimatic conditions were described by using a metabarcoding strategy targeting ITS2 and 16S barcodes. Results: A total of 11,974 fungal and 12,010 bacterial amplicon sequence variants (ASVs) were obtained, revealing rich and diverse microbial communities associated with different cork oak forests. Bioclimate was described as the major factor influencing variability in these communities (or bioclimates/cork oak forest for fungal community), followed by boron and granulometry. Also, pH explained variation of fungal communities, while C:N ratio contributed to bacterial variation. Fungal and bacterial biomarker genera for specific bioclimates were described. Their co-occurrence network revealed the existence of a complex and delicate balance among microbial communities. Conclusions: The findings revealed that bacterial communities are more likely to be affected by different edaphoclimatic conditions than fungal communities, also predicting a higher impact of climate change on bacterial communities. The integration of cork oak fungal and bacterial microbiota under different bioclimates could be further explored to provide information about useful interactions for increasing cork oak forest sustainability in a world subject to climate changes.This work was supported 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 POCI-01-0145- FEDER-028635, as well as for financial support to BioISI (UIDB/04046/2020), CBMA (UIDB/04050/2020) and CIMO (UID/AGR/00690/2020) by national funds by FCT/MCTES/PIDDAC. D. Costa thanks FCT for PhD grant SFRH/ BD/120516/2016 and COVID/BD/151779/2021.info:eu-repo/semantics/publishedVersio

Macrofungi associated with sweet chestnut: a source of income for rural populations in Northeast of Portugal

European chestnut tree (Castanea sativa Mill.) has a great economic interest for wood and fruit production in northeast of Portugal. Over the last decade another important income associated with this culture has emerged - the collection and commercialization of wild edible mushrooms growing in chestnut orchards. Actually, despite the low knowledge of their biodiversity, ecology or sustainable management in those regions, mushrooms harvesting have been increasing, mainly due to their economic importance to local populations. In order to define the sustainability of this mycological resource, the study of diversity and production of macrofungi associated with chestnut tree was carried out. Commercial harvesting of edible mushrooms as a complementary income to local populations, was also evaluated, through chestnut associated mushroom production. The study was carried out from 2002 to 2005, in a non-tilled C. sativa orchard located in Oleiros – Bragança (Northeast of Portugal) (29T PG 80 9 36 UTM, 915 m above sea level), at Natural Park of Montesinho. Five quadrate plots of 100 m2 each were selected in the chestnut orchard, and all the sporocarps were weekly (autumn and spring) or biweekly (winter and summer) collected. No assessments were undertaken during January, February, March and August, since in this period few macrofungi produce sporocarps in Bragança. Sporocarps were identified by genera or species following standard procedures for taxonomic identification. For determining production, sporocarps were dried at 30ºC, for 72 h, and weighed (dw). Meteorological data was obtained from the weather station at Campus of Escola Superior Agrária de Bragança, located approximately 6 km of the studied C. sativa orchard. The diversity of macrofungi species was expressed by species richness (S), Simpson´s diversity index (D), total abundance (N) and relative abundance. The production of macrofungi (dry weight per hectare) was expressed for i) total species; ii) mycorrhizal and non-mycorrhizal species; iii) and edible and non-edible species. The economic income was only evaluated for the edible species sold in the local markets, namely Amanita caesarea, Boletus edulis, Boletus aereus, Cantharellus cibarius, Hydnum rufescens and Calocybe gambosa (Garcia et al., 2006). The value of mushrooms production was estimated taking into account the average current price of these species in the region in 2003. During the period of studies, 73 macrofungal species belonging to 16 family and 23 genera were identified. The greatest number of species belonged to the genera Russula (12), Inocybe (9), Lactarius (7), Tricholoma (6), Boletus (5), Cortinarius (5) and Amanita (5). The cumulative number of species, over the four successive years, revealed that 80.5% of macrofungal species that probably exist in this habitat were surveyed. The harvestings from 2002 and 2003 has contributed mostly to the number of species collected. Actually, the number of macrofungal species attained a maximum (56 species) in 2002 and a minimum (12 species) in 2005. Also, Simpson´s diversity index was significantly higher in 2002 (8.1 ± 0.98) than in the other years (3.9 ± 0.51 in 2003; 1.5 ± 0.15 in 2004; 1.1 ± 0.06 in 2005). About 46% of the macrofungal species has occurred in a single year. Only about 4% of the species has occurred in all de 4-years study, 25% in two years and 25% in three years. Fruiting phenology of macrofungal species and number of carpophores showed a bimodal pattern in each year, with one peak from late May to June (Spring season) and a more intense peak from September to early December (Autumn season). The greatest number of species (85% of the total registered) has occurred during Autumn season and only 11 species have occurred in both fruiting seasons (Spring and Autumn). A total of 2677 carpophores were harvested during the time course of this study. The higher number of carpophores was from the species Laccaria laccata (324), Hebeloma crustuliniforme (308) and Inocybe geophylla (303), which were also the most frequent species over the 4-year study period besides Russula lutea and Cortinarius helobius. The maximum number of carpophores was observed in 2003 (1514 carpophores) and the minimum in 2005 (138 carpophores). Also, the species richness and abundance were much lower in 2004 and especially in 2005, than in 2002 and 2003. These results were probably the effect from a severe drought period observed during all 2004 and 2005 in the Northeast of Portugal. The distribution of species by trophy groups revealed a dominance of mycorrhizal species (82%). About 49% of the identified species were edible and 29% were non-edible, the remaining being of unknown edibility. From the 36 identified edible species, only six are currently available in local markets. The total carpophores yield during the 4-year study period attained 67.6 kg dw/ha, from which 64.0 kg dw/ha were mycorrhizal species and 50.7 kg dw/ha were edible species. Lactarius piperatuts, Russula delica and Boletus edulis were the species that contributed most to the total yield (11.0 kg dw/ha, 8.0 kg dw/ha and 6.2 kg dw/ha, respectively). As observed for species richness and abundance, also the macrofungi yield reached a maximum (44.0 kg dw/ha) in 2002 and a minimum (2.3 kg dw/ha) in 2005. Only 20% of the total dry weight production of edible species refers to current available mushroom in local markets. Assuming the average current price of these species in the region in 2003, the total income associated with these species was 134 €/ha and per year: A. caesarea (3.5 €/ha), B. edulis (59.4 €/ha), B. aereus (18.2 €/ha), C. cibarius (39.8 €/ha), H. rufescens (3.7 €/ha) and C. gambosa (9.0 €/ha). These results suggest that commercial harvesting of these edible mushrooms could be an additional source of income for chestnut producers with significant contribution to regional economies. However, over picking has to be avoided, as conservation and management strategies are required. In fact, it is important that these habitats will be managed under an ecosystem management philosophy that entails multiple-use. For this purpose it is still necessary to study mushroom harvesting effects in the ecosystem and the effects of management practices (namely tillage or non tillage) on the occurrence, production and reproduction of mushrooms. To improve mushroom management it will also necessary to understand their biology and ecology, and perform mushrooms surveys over multiple fruiting seasons.European chestnut tree (Castanea sativa Mill.) has a great economic interest for wood and fruit production in northeast of Portugal. Over the last decade another important income associated with this culture has emerged - the collection and commercialization of wild edible mushrooms growing in chestnut orchards. Actually, despite the low knowledge of their biodiversity, ecology or sustainable management in those regions, mushrooms harvesting have been increasing, mainly due to their economic importance to local populations. In order to define the sustainability of this mycological resource, the study of diversity and production of macrofungi a ssociated with chestnut tree was carried out.The authors ore grateful to AGRO Project 689 for financial support of this work

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

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

Endo- and epiphytic fungal communities of olive twigs is influenced by cultivar and olive knot infection

Olive tree phyllosphere is colonized by a diverse microbial assemblage that may interact with pathogenic fungi, making them potential candidates for disease supression. Olive knot (OK) is a disease caused by the bacterium Pseudomonas savastanoi pv. savastanoi (Psv.) with significant economic losses in olive tree (Olea europaea L.). With this work we want to evaluate the effect of cultivar and OK infection on both endo- and epiphytic fungal community inhabiting olive twig tissues. For this, fungal composition and diversity was assessed in both asymptomatic and OK-symptomatic twigs of tree co-occurring olive cultivars with different susceptibilities to OK disease. The identification of isolated species was performed by ITS rDNA sequencing. The cultivar and OK infection were important in shaping both endophytic and epiphytic fungal communities. Fungal community composition was found to differ significantly (p=0.005) between olive tree cultivars, being Nectriaceae the dominant family in cvs. Cobrançosa and Verdeal Transmontana, whereas Pleosporaceae was dominant in the cv. Madural. Epiphytic and endophytic fungal communities also differed in size and in composition in asymptomatic and OK-symptomatic twigs, for the three cultivars. In general, asymptomatic twigs were more diverse and rich (up to 1.4-fold) when compared to OK-symptomatic twigs. Among the species identified in the asymptomatic tissues, Cladosporium cladosporioides and Chromelosporium carneum were the most frequently isolated within epiphytic and endophytic community, respectively; whereas in the OK-symptomatic tissues Cladosporium sp. and Fusarium lateritium were the most frequently isolated within epiphytic and endophytic community, respectively. According to indicator species analysis C. carneum, Pyronema dosmesticum and Phoma aloes (IndVal up to 0.56) may be promising species in the OK suppression. Better acknowledgement should be developed in order to uncover their role on plant health.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. T. Gomes thanks FCT, POPH-QREN and FSE for PhD SFRH/BD/98127/2013 Grantinfo:eu-repo/semantics/publishedVersio

Phylogenetic analysis and genetic diversity of the xylariaceous ascomycete Biscogniauxia mediterranea from cork oak forests in different bioclimates

Cork oak is a tree species with ecological importance that contributes to economic and social development in the Mediterranean region. Cork oak decline is a major concern for forest sustainability and has negative impacts on cork oak growth and production. This event has been increasingly reported in the last decades and seems to be related with climate changes. Biscogniauxia mediterranea is an endophytic fungus of healthy cork oak trees that turns into a pathogen in trees weaken by environmental stress. Understanding the drivers of B. mediterranea populations diversity and differentiation is expected to allow a better control of cork oak decline and preserve forest sustainability. Endophyte isolates from different cork oak forests were identified as B. mediterranea and their genetic diversity was evaluated using phylogenetic and microsatellite-primed PCR analyses. Genetic diversity and variability of this fungus was correlated with environmental/phytosanitary conditions present in forests/trees from which isolates were collected. High genetic diversity and variability was found in B. mediterranea populations obtained from different forests, suggesting some degree of isolation by distance. Bioclimate was the most significant effect that explained the genetic variability of B. mediterranea, rather than precipitation or temperature intensities alone or disease symptoms. These findings bring new implications for the changing climate to cork oak forests sustainability, cork production and quality.This work was supported 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 POCI-01-0145-FEDER-028635, as well as for financial support to BioISI (UIDB/04046/2020), CBMA (UIDB/04050/2020), and CIMO (UID/AGR/00690/2020) by national funds by FCT/MCTES/PIDDAC. D. Costa thanks FCT for PhD grant SFRH/BD/120516/2016.info:eu-repo/semantics/publishedVersio

Ability of fungi isolated from olive tree cultivars with different susceptibilities to olive knot to inhibit Pseudomonas savastanoi pv. savastanoi

The Olive Knot is distributed in olive-growing regions worldwide. This disease is caused by the Pseudomonas savastanoi pv. sovastonoí (Psv), a bacterium that produces galls especially on stems and twigs of olive tree. The use of biological control agents (BCAs)to control plant diseases have gained increasing attention during the last decades, due the negative impact of pesticides on environment and human health. This strategy can offer an alternative in order to achieve a sustainable and environ mentally friendly agriculture. The knowledge of fungal communities inhabiting the phyllosphere of olive tree and the type of interaction that they established with the host plant is essential to explore the use of these microorganisms as BCAs against Olive Knot. Previously, we have characterized the fungal communities inhabiting either the surface (epiphytes) or internal tissues of twigs (endophytes)from olive tree cultivars with different susceptibilities to Olive Knot (Cobrançosa moderately tolerant; Verdeal Transmontana susceptible). In this work, 43 endo- and epiphytes of this fungal community were screened for the suppression of Psv, and several mechanisms behind this activity were also studied by evaluating lytic enzymes production. The interspecific interaction was assessed on two different culture media, potato dextrose agar (PDA)and olive leaf twigs extract (OlTE), with agar overlays and by measuring the zone of growth inhibition. Lytic enzymes were evaluated qualitatively. In general, 95% of the fungal species tested inhibited significantly Psv and above an inhibition index (11) threshold of 1.5, being Dothiorella iberica, Rhinocladiella similis, Phoma aloes and Quambalaria cyanescens the species thatexhibited the maximum values (11 ranging from 31 to 19).The inhibition was observed to be affected by growth medium and correlated with the cultivar from which fungi were isolated. In OlTE was observed higher growth inhibition (up to 1.7 fold) and number of species with 11> 5 (up to 1.9 fold), compared to medium without host plant extracts. Similarly, fungal species isolated from the tolerant cultivar Cobrançosa showed higher capacity to inhibit Psv (up to 1.3 fold) when compared to isolates from cv.Verdeal Transmontana. This effect was noticed either for endophytes or epiphytes. The production of lipase, protease, amylase, and cellulase by fungi contributed to the antagonistic activity against Psv, whose production was influenced by olive tree extracts. Overall, results revealed that intimate physical interaction between fungal endophyte/epiphyte with Psvin the presence of olive tree extracts leads to the production of fungal secondary metabolite compounds that inhibit the pathogen growth, which were otherwise not produced. The potential of these fungal species as biological control agents should be considered and further evaluated under "in vivo" conditions.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.This work is funded by FEDERfunds through COMPETE(Programa Operacional Factores de Competitividade) and by national funds by FCT(Fundação para a Ciência ea Tecnologia) inthe framework of the project EXCL/AGR-PRO!0591/2012.T. Gomes thanks FCT, POPH-QRENand FSE for PhD SFRH/BD/98127/2013 Grant.info:eu-repo/semantics/publishedVersio