Sustainable control of pea bacterial blight : approaches for durable genetic resistance and biocontrol by endophytic bacteria

Key-words: bacterial blight, biological control, biodiversity, endophytic bacteria, L-form, pea, PDRl retrotransposon, Pisum sativum, Pisum abyssinicum, Pseudomonas syringae pv. pisi, race specific resistance, race non-specific resistance, Spanish landraces.Pea bacterial blight (Pseudomonas syringae pv. pisi) occurs worldwide and can cause severe damage under cool and wet conditions particularly at the seedling stage in wintersown crops. Seven Ps. syr. pv. pisi races are currently recognized. There are no resistant cultivars to race 6, which is becoming increasingly important. Current disease control measures include disease avoidance through seed testing and the deployntent of resistant cultivars with race specific resistance gene(s). In the present study two novel control measures were investigated with the potential for integration to give a durable and sustainable disease control. The first was breeding for resistance based on race non-specific resistance present in Pisum abyssinicum, which confers resistance to all races, including race 6. Its mode of inheritance was investigated through a crossing programme with Pisum sativum cultivars. Resistance was controlled by a major recessive gene and a number of modifiers. Progenies of crosses between resistant F5 populations and commercial cultivars are now available. Molecular markers for race non-specific resistance based on a pea retrotransposon marker system were developed. It is suggested that the combination of race specific and race non-specific resistance provides the optimal genetic background for the maximum expression of resistance to all races of the pathogen in all plant parts and under field conditions. The second measure was biological control by endophytic bacteria. Studies on the ecology of endophytic bacteria in pea and identification of efficient indigenous colonizers for potential application in biocontrol have been made. Endophytic population levels were in the range 10 3 -10 6 CFU/g fresh tissue in roots and stems. There was a predominance of Gram-negative bacteria, particularly Pseudomonas sp. and Pantoea agglomerans. Arthrobacter sp. and Curtobacterium sp. were the main Gram-positive bacteria. Factors such as soil type, plant genotype and crop growth stage may significantly influence the diversity and population levels of endophytic bacteria. Future research should focus on the combination and testing of elite breeding lines with selections of disease suppressive endophytic isolates under a variety of field conditions in order to obtain an efficient and durable performance in commercial agriculture.</p

Root infection of canker pathogens, Fusarium circinatum and Diplodia sapinea, in asymptomatic trees in Pinus radiata and Pinus pinaster plantations

[EN] The existence of a latent stage within host tissue of the pine pathogens Fusarium circinatum and Diplodia sapinea, the causal agents of pitch canker and shoot blight disease respectively, has previously been cited. However, studies on this cryptic phase in each disease lifecycle has only been focused on the host aerial parts but not on the roots. Therefore, our objective was to analyze the presence of both pathogens in roots of non-symptomatic mature trees in plantations where the pathogens are known to be causing canker symptoms. For that, we sampled roots from ten non-symptomatic and ten symptomatic trees in three Pinus radiata and one Pinus pinaster plantations in Basque Country, Spain. Both pathogens were isolated from roots of non-symptomatic trees in a higher frequency than from roots of symptomatic trees, 23.3% and 6.6% respectively for D. sapinea and 16.6% and 3.3% respectively for F. circinatum. Neither pathogens was detected in the P. pinaster plantation. The two pathogens were never isolated from the same tree. A high molecular variability was observed for D. sapinea isolates with six different haplotypes and two mating types for the eleven characterized isolates, but only one haplotype and mating type was found for F. circinatum, with all isolates of both fungi being proved pathogenic. These results evidence the importance root infection may have in the disease lifecycle and, therefore, disease management.We acknowledge Maria Teresa Morales Clemente for her excellent technical assistance. Laura Hernandez-Escribano was supported by a fellowship from INIA (FPI-INIA). Financial support for this research was provided by project RTA2013-00048-C03-01, RTA2017-00063-C04-01 and C04-03 (National Progamme I + D + I, INIA, Spain) and the Project Healthy Forest LIFE14 ENV/ES/000179. This article is-based upon work from COST Action FP1406, Pine pitch canker-strategies for management of Gibberella circinata in greenhouses and forests (PINESTRENGTH), supported by COST (European Cooperation in Science and Technology).Hernandez-Escribano, L.; Iturritxa, E.; Aragonés, A.; Mesanza, N.; Berbegal Martinez, M.; Raposo, R.; Elvira-Recuenco, M. (2018). Root infection of canker pathogens, Fusarium circinatum and Diplodia sapinea, in asymptomatic trees in Pinus radiata and Pinus pinaster plantations. Forests. 9(3):1-15. https://doi.org/10.3390/f9030128S11593Nirenberg, H. I., & O’Donnell, K. (1998). New Fusarium Species and Combinations within the Gibberella fujikuroi Species Complex. Mycologia, 90(3), 434. doi:10.2307/3761403Phillips, A. J. L., Alves, A., Abdollahzadeh, J., Slippers, B., Wingfield, M. J., Groenewald, J. Z., & Crous, P. W. (2013). The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology, 76, 51-167. doi:10.3114/sim0021Wingfield, M. 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Latent Infection of Austrian and Scots Pine Tissues by Sphaeropsis sapinea. Plant Disease, 85(10), 1107-1112. doi:10.1094/pdis.2001.85.10.1107Flowers, J., Hartman, J., & Vaillancourt, L. (2003). Detection of Latent Sphaeropsis sapinea Infections in Austrian Pine Tissues Using Nested-Polymerase Chain Reaction. Phytopathology®, 93(12), 1471-1477. doi:10.1094/phyto.2003.93.12.1471Smith, H., Wingfied, M. ., & Coutinho, T. . (2002). The role of latent Sphaeropsis sapinea infections in post-hail associated die-back of Pinus patula. Forest Ecology and Management, 164(1-3), 177-184. doi:10.1016/s0378-1127(01)00610-7Vujanovic, V., St-Arnaud, M., & Neumann, P.-J. (2000). Susceptibility of cones and seeds to fungal infection in a pine (Pinus spp.) collection. Forest Pathology, 30(6), 305-320. doi:10.1046/j.1439-0329.2000.00211.xBihon, W., Slippers, B., Burgess, T., Wingfield, M. J., & Wingfield, B. D. (2010). Sources of Diplodia pinea endophytic infections in Pinus patula and P. radiata seedlings in South Africa. Forest Pathology, 41(5), 370-375. doi:10.1111/j.1439-0329.2010.00691.xFABRE, B., PIOU, D., DESPREZ-LOUSTAU, M.-L., & MARÇAIS, B. (2011). Can the emergence of pine Diplodia shoot blight in France be explained by changes in pathogen pressure linked to climate change? Global Change Biology, 17(10), 3218-3227. doi:10.1111/j.1365-2486.2011.02428.xSwett, C. L., Kirkpatrick, S. C., & Gordon, T. R. (2016). Evidence for a Hemibiotrophic Association of the Pitch Canker Pathogen Fusarium circinatum with Pinus radiata. Plant Disease, 100(1), 79-84. doi:10.1094/pdis-03-15-0270-reMartín-Rodrigues, N., Sanchez-Zabala, J., Salcedo, I., Majada, J., González-Murua, C., & Duñabeitia, M. K. (2015). New insights into radiata pine seedling root infection byFusarium circinatum. Plant Pathology, 64(6), 1336-1348. doi:10.1111/ppa.12376Swett, C. L., & Gordon, T. R. (2016). Exposure to a pine pathogen enhances growth and disease resistance inPinus radiataseedlings. Forest Pathology, 47(1), e12298. doi:10.1111/efp.12298Stanosz, G. R., Blodgett, J. T., Smith, D. R., & Kruger, E. L. (2001). Water stress and Sphaeropsis sapinea as a latent pathogen of red pine seedlings. New Phytologist, 149(3), 531-538. doi:10.1046/j.1469-8137.2001.00052.xBihon, W., Burgess, T., Slippers, B., Wingfield, M. J., & Wingfield, B. D. (2011). Distribution of Diplodia pinea and its genotypic diversity within asymptomatic Pinus patula trees. Australasian Plant Pathology, 40(5), 540-548. doi:10.1007/s13313-011-0060-zAegerter, B. J., & Gordon, T. R. (2006). Rates of pitch canker induced seedling mortality among Pinus radiata families varying in levels of genetic resistance to Gibberella circinata (anamorph Fusarium circinatum). Forest Ecology and Management, 235(1-3), 14-17. doi:10.1016/j.foreco.2006.07.011Nirenberg, H. I. (1981). A simplified method for identifying Fusarium spp. occurring on wheat. Canadian Journal of Botany, 59(9), 1599-1609. doi:10.1139/b81-217Slippers, B., Crous, P. W., Denman, S., Coutinho, T. A., Wingfield, B. D., & Wingfield, M. J. (2004). Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified asBotryosphaeria dothidea. Mycologia, 96(1), 83-101. doi:10.1080/15572536.2005.11833000Alves, A., Linaldeddu, B. T., Deidda, A., Scanu, B., & Phillips, A. J. L. (2014). The complex of Diplodia species associated with Fraxinus and some other woody hosts in Italy and Portugal. Fungal Diversity, 67(1), 143-156. doi:10.1007/s13225-014-0282-9Hyde, K. D., Nilsson, R. H., Alias, S. A., Ariyawansa, H. A., Blair, J. E., Cai, L., … Zhou, N. (2014). One stop shop: backbones trees for important phytopathogenic genera: I (2014). Fungal Diversity, 67(1), 21-125. doi:10.1007/s13225-014-0298-1Dissanayake, A. (2016). 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Phytopathology®, 103(8), 851-861. doi:10.1094/phyto-11-12-0281-rIturritxa, E., Ganley, R. J., Wright, J., Heppe, E., Steenkamp, E. T., Gordon, T. R., & Wingfield, M. J. (2011). A genetically homogenous population of Fusarium circinatum causes pitch canker of Pinus radiata in the Basque Country, Spain. Fungal Biology, 115(3), 288-295. doi:10.1016/j.funbio.2010.12.014Elvira-Recuenco, M., Iturritxa, E., Majada, J., Alia, R., & Raposo, R. (2014). Adaptive Potential of Maritime Pine (Pinus pinaster) Populations to the Emerging Pitch Canker Pathogen, Fusarium circinatum. PLoS ONE, 9(12), e114971. doi:10.1371/journal.pone.0114971Garbelotto, M., Smith, T., & Schweigkofler, W. (2008). Variation in Rates of Spore Deposition of Fusarium circinatum, the Causal Agent of Pine Pitch Canker, Over a 12-Month-Period at Two Locations in Northern California. Phytopathology®, 98(1), 137-143. doi:10.1094/phyto-98-1-0137Serra-Varela, M. J., Alía, R., Pórtoles, J., Gonzalo, J., Soliño, M., Grivet, D., & Raposo, R. (2017). Incorporating exposure to pitch canker disease to support management decisions of Pinus pinaster Ait. in the face of climate change. PLOS ONE, 12(2), e0171549. doi:10.1371/journal.pone.0171549Hernandez-Escribano, L., Iturritxa, E., Elvira-Recuenco, M., Berbegal, M., Campos, J. A., Renobales, G., … Raposo, R. (2018). Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum. Fungal Ecology, 32, 65-71. doi:10.1016/j.funeco.2017.12.001Smith, H., Wingfield, M. J., Coutinho, T. A., & Crous, P. W. (1996). Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany, 62(2), 86-88. doi:10.1016/s0254-6299(15)30596-2Santini, A., Pepori, A., Ghelardini, L., & Capretti, P. (2008). Persistence of some pine pathogens in coarse woody debris and cones in a Pinus pinea forest. Forest Ecology and Management, 256(3), 502-506. doi:10.1016/j.foreco.2008.05.010Oblinger, B. W., Smith, D. R., & Stanosz, G. R. (2011). Red pine harvest debris as a potential source of inoculum of Diplodia shoot blight pathogens. Forest Ecology and Management, 262(4), 663-670. doi:10.1016/j.foreco.2011.04.038Eyles, A., Bonello, P., Ganley, R., & Mohammed, C. (2009). Induced resistance to pests and pathogens in trees. New Phytologist, 185(4), 893-908. doi:10.1111/j.1469-8137.2009.03127.xJunker, C., Draeger, S., & Schulz, B. (2012). A fine line – endophytes or pathogens in Arabidopsis thaliana. Fungal Ecology, 5(6), 657-662. doi:10.1016/j.funeco.2012.05.002Flowers, J. L., Hartman, J. R., & Vaillancourt, L. J. (2006). Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots. Forest Pathology, 36(6), 447-459. doi:10.1111/j.1439-0329.2006.00473.

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems

[EN] Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9-SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.This research was funded by INIA, grant number: RTA 2017-00063-C04-03, LIFE programme, grant number: LIFE14 ENV/ES/000179 and by the Basque Government, grant number FUNGITRAP 19-00031. Red pine cone collection in New England and pathogen isolation was funded by USDA Forest Service.Aragonés, A.; Manzanos, T.; Stanosz, G.; Munck, IA.; Raposo, R.; Elvira-Recuenco, M.; Berbegal Martinez, M.... (2021). Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems. Microorganisms. 9(12):1-17. https://doi.org/10.3390/microorganisms9122565S11791

Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum

[EN] Fusarium circinatum was recently detected as an endophyte in grasses causing no apparent damage. Our goal was to describe the endophytic colonization of herbaceous host plants growing in a plantation of Pinus radiata with symptoms of pitch canker disease, which may act as a reservoir of inoculum. We detected the fungus in five species of dicot families (Asteraceae, Lamiaceae, Rosaceae), in addition to two species in the Poaceae. The fungus was found in the aerial part of non-symptomatic hosts, so we describe E circinatum as an endophyte that is mainly transmitted by spores through the air. It was also detected in Hypochaeris radicata seeds, suggesting the potential occurrence of vertical transmission. An analysis of microsatellite markers showed a unique haplotype regardless of whether the isolates' origin was pine cankers or non-symptomatic herbaceous plants. Thus, the same genotype can adopt a pathogenic or endophytic lifestyle. We conclude that non -symptomatic plants can act as reservoirs of inoculum: pine seedlings can be infeded from senescent tissue of non-symptomatic hosts colonized by the fungus. (C) 2017 Elsevier Ltd and British Mycological Society. All rights reserved.We acknowledge Maite Morales Clemente for her excellent technical assistance and Inigo Zabalgogeazcoa for his helpful suggestions. Laura Hernandez was supported by a fellowship from INIA (FPI-INIA). Financial support for this research was provided by projects RTA2012-00015-C02-01 and RTA2013-00048-C03-01 (Programa Estatal I + D + i, INIA, Spain).Hernandez-Escribano, L.; Iturritxa, E.; Elvira-Recuenco, M.; Berbegal Martinez, M.; Campos, J.; Renobales, G.; García, I.... (2018). Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum. Fungal Ecology. 32:65-71. https://doi.org/10.1016/j.funeco.2017.12.001S65713

Pine Pitch Canker and Insects: Regional Risks, Environmental Regulation, and Practical Management Options

Producción CientíficaPine pitch canker (PPC), caused by the pathogenic fungus Fusarium circinatum (Nirenberg and O’ Donnell), is a serious threat to pine forests globally. The recent introduction of the pathogen to Southern Europe and its spread in Mediterranean region is alarming considering the immense ecological and economic importance of pines in the region. Pines in forests and nurseries can be infected, resulting in severe growth losses and mortality. The pathogen is known to spread in plants for planting and in seeds, and results from recent studies have indicated that F. circinatum may also spread through phoretic associations with certain insects. With this review, we aim to expand the current understanding of the risk of insect-mediated spread of PPC in different parts of Europe. Through the joint action of a multinational researcher team, we collate the existing information about the insect species spectrum in different biogeographic conditions and scrutinize the potential of these insects to transmit F. circinatum spores in forests and nurseries. We also discuss the impact of environmental factors and forest management in this context. We present evidence for the existence of a high diversity of insects with potential to weaken pines and disseminate PPC in Europe, including several common beetle species. In many parts of Europe, temperatures are projected to rise, which may promote the activity of several insect species, supporting multivoltinism and thus, further amplifying the risk of insect-mediated dissemination of PPC. Integrated pest management (IPM) solutions that comply with forest management practices need to be developed to reduce this risk. We recommend careful monitoring of insect populations as the basis for successful IPM. Improved understanding of environmental control of the interaction between insects, the pathogen, and host trees is needed in order to support development of bio-rational strategies to safeguard European pine trees and forests against F. circinatum in future.European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH)Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R)Portuguese Foundation for Science and Technology (contract IF/00471/2013/CP1203/CT0001)Russian Foundation for Basic Research (grant 17-04-01486)Saint Petersburg State Polytechnical University (project 2019-0420

Phyllosphere microbiology with special reference to diversity and plant genotype

The phyllosphere represents the habitat provided by the aboveground parts of plants, and on a global scale supports a large and complex microbial community. Microbial interactions in the phyllosphere can affect the fitness of plants in natural communities, the productivity of agricultural crops, and the safety of horticultural produce for human consumption. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonists, which is influenced by numerous environmental factors in addition to leaf physico-chemical properties. The recent use of culture-independent techniques has demonstrated considerable previously unrecognized diversity in phyllosphere bacterial communities. Furthermore, there is significant recent evidence that plant genotype can play a major role in determining the structure of phyllosphere microbial communities. The main aims of this review are: (i) to discuss the diversity of phyllosphere microbial populations; (ii) to consider the processes by which microbes colonize the phyllosphere; (iii) to address the leaf characteristics and environmental factors that determine the survival and growth of colonists; (iv) to discuss microbial adaptations that allow establishment in the phyllosphere habitat and (v) to evaluate evidence for plant genotypic control of phyllosphere communities. Finally, we suggest approaches and priority areas for future research on phyllosphere microbiology

The increasing threat to European forests from the invasive foliar pine pathogen, Lecanosticta acicola

European forests are threatened by increasing numbers of invasive pests and pathogens. Over the past century, Lecanosticta acicola, a foliar pathogen predominantly of Pinus spp., has expanded its range globally, and is increasing in impact. Lecanosticta acicola causes brown spot needle blight, resulting in premature defoliation, reduced growth, and mortality in some hosts. Originating from southern regions of North American, it devastated forests in the USA's southern states in the early twentieth century, and in 1942 was discovered in Spain.Derived from Euphresco project 'Brownspotrisk,' this study aimed to establish the current distribution of Lecanosticta species, and assess the risks of L. acicola to European forests. Pathogen reports from the literature, and new/ unpublished survey data were combined into an open-access geo-database (http://www.portaloff orestpathology.com), and used to visualise the pathogen's range, infer its climatic tolerance, and update its host range. Lecanosticta species have now been recorded in 44 countries, mostly in the northern hemisphere. The type species, L. acicola, has increased its range in recent years, and is present in 24 out of the 26 European countries where data were available. Other species of Lecanosticta are largely restricted to Mexico and Central America, and recently Colombia.The geo-database records demonstrate that L. acicola tolerates a wide range of climates across the northern hemisphere, and indicate its potential to colonise Pinus spp. forests across large swathes of the Europe. Pre-liminary analyses suggest L. acicola could affect 62% of global Pinus species area by the end of this century, under climate change predictions.Although its host range appears slightly narrower than the similar Dothistroma species, Lecanosticta species were recorded on 70 host taxa, mostly Pinus spp., but including, Cedrus and Picea spp. Twenty-three, including species of critical ecological, environmental and economic significance in Europe, are highly susceptible to L. acicola, suffering heavy defoliation and sometimes mortality. Variation in apparent susceptibility between reports could reflect variation between regions in the hosts' genetic make-up, but could also reflect the signif-icant variation in L. acicola populations and lineages found across Europe. This study served to highlight sig-nificant gaps in our understanding of the pathogen's behaviour.Lecanosticta acicola has recently been downgraded from an A1 quarantine pest to a regulated non quarantine pathogen, and is now widely distributed across Europe. With a need to consider disease management, this study also explored global BSNB strategies, and used Case Studies to summarise the tactics employed to date in Europe