Plant-associated microbiota as a source of antagonistic bacteria against the phytopathogen Erwinia amylovora

Control of bacterial plant diseases is a major concern, as they affect economically important species and spread easily, such as the case of fire blight of rosaceous caused by Erwinia amylovora. In the search for alternatives to the use of agrochemicals and antibiotics, this work presents a screening of natural bacterial antagonists of this relevant and devastating phytopathogen. We recovered bacterial isolates from different plant tissues and geographical origins and then selected those with the strongest ability to reduce fire blight symptoms ex vivo and remarkable in vitro antagonistic activity against E. amylovora. None of them elicited a hypersensitivity reaction in tobacco leaves, most produced several hydrolytic enzymes and presented other biocontrol and/or plant growth-promoting activities, such as siderophore production and phosphate solubilization. These isolates, considered as biocontrol candidates, were identified by 16S rRNA sequencing as Pseudomonas rhizosphaerae, Curtobacterium flaccumfaciens, Enterobacter cancerogenus, Pseudomonas azotoformans, Rosenbergiella epipactidis and Serratia plymuthica. This is the first time that the last five bacterial species are reported to have biocontrol potential against E. amylovora

Root-knot nematodes: abundance in organic farming, differentiation among populations, microbes attached to juveniles in soil, and bacterial antagonists

In zwei umfassenden Untersuchungen im Herbst 2009 und 2011 wurde die Verbreitung und die Abundanz pflanzenparasitärer Nematoden an den verschiedenen Feldfrüchten in einer Bio-Farm in Ägypten erfasst. Insgesamt wurden elf Genera gefunden, von denen Wurzelgallen-Nematoden (Meloidogyne spp.) in beiden Untersuchungen am häufigsten und mit der höchsten Anzahl in den Proben gefunden wurde. Weitere oft gefundene Genera waren Tylenchorhynchus, Rotylenchulus, Helicotylenchus und Pratylenchus. Davon ausgehend beschäftigten sich die weiteren Arbeiten mit Meloidogyne, und zwar mit der Differenzierung von Populationen, der Anheftung von Mikroorganismen an die Juvenile (J2) im Boden, und der biologischen Kontrolle durch Bakterienstämme, die als Antagonisten von pilzlichen Pathogenen bekannt sind. Die untersuchten Populationen und/oder Rassen von Meloidogyne incognita zeigten phänotypische Unterschiede in ihren Vermehrungsmustern an einem Set von Wirtspflanzen. Damit einhergehend konnten auch genetische Unterschiede anhand einer neu entwickelten Methode zur Amplifikation des Pathogenitätsgens msp1 und der elektrophoretischen Auftrennung der PCR-Produkte von Genvarianten im Denaturierungsgradienten (DGGE) nachgewiesen werden. Für drei Ackerböden aus verschiedenen Regionen in Deutschland wurde unterschiedliche Suppressivität ihrer mikrobiellen Gemeinschaften gegen Meloidogyne hapla im Gewächshaus gezeigt. Mit Kultivierungs-unabhängigen Methoden wurde untersucht, welche Mikroorganismen an die J2 in den Böden anheften, um die zu identifizieren, die mit J2 im suppressivsten der drei Böden spezifisch interagierten (Kleinwanzleben). Die Mikroorganismen, die an den J2 nach Inkubation im Boden haften blieben, unterschieden sich zwischen den drei Böden. In PCR-DGGE Fingerprints von ITS-Fragmenten bzw. 16S rRNA Genen wurden viele Pilze und Bakterien detektiert, die an den J2 aber nicht im umgebenden Boden abundant waren. Während sich einige davon in allen drei Böden an den J2 anreicherten, waren andere spezifisch für einen Bodentyp. Mittels Pyrosequenzierung von 16S rRNA Gen-Amplikons konnten die mit J2 im suppressivsten Boden assoziierten abundantesten Bakterienarten beschrieben werden. Viele davon waren verwandt mit infektiösen Arten wie Shigella spp., während Malikia spinosa und Rothia amarae am häufigsten detektiert wurden. Krankheitskomplexe aus Nematode und Pilz können erhebliche synergistische Ertragsverluste verursachen. Bakterienstämme, die als Antagonisten von phytopathogenen Pilzen bekannt sind, wurden auf ihr Potential zur biologischen Kontrolle von M. incognita untersucht. Sameninokulation führte bei den meisten Stämmen zu einer signifikanten Reduktion der Vermehrung des Nematoden an Tomatenwurzeln. Für die drei besten Isolate, Bacillus subtilis Sb4-23, Mc2-Re2 und Mc5- Re2, wurden die zugrundeliegenden Mechanismen untersucht. Die Stämme konnten den Nematoden sowohl direkt durch Metabolite beeinträchtigen, die im Kulturüberstand zu finden sind, als auch indirekt über Induktion systemischer Resistenz der Pflanze. Im experimentellen Vergleich der direkten und Pflanzenvermittelten Effekte zeigte sich, dass letzteres der dominierende Kontrollmechanismus dieser Antagonisten ist. Zusammen genommen könnten diese Befunde als Basis für eine verbesserte Strategie zum integrierten Management von Wurzelgallen-Nematoden im biologischen Landbau dienen.Two surveys were conducted to determine the frequency and abundance of plantparasitic nematodes associated with different crops at an organic farm in Egypt, during autumn 2009 and 2011. Eleven genera of plant-parasitic nematodes were detected. Root-knot nematodes (Meloidogyne spp.) showed the highest abundance and frequency of all plant-parasitic nematodes during the two surveys. Commonly detected genera were Tylenchorhynchus, Rotylenchulus, Helicotylenchus and Pratylenchus. Further studies on Meloidogyne were carried out with regard to discrimination among populations, attachment of microbes to juveniles (J2) in soil, and biocontrol through bacterial strains which were known as antagonists of fungal pathogens. Meloidogyne incognita populations and/or races that showed differential pattern of reproduction on a set of host plants, could be differentiated based on a newly developed PCR-DGGE system to electrophorecically separate variants of the pathogenicity gene msp1. Three arable soils from different regions of Germany were shown to vary in the suppressiveness of their indigenous microbial communities against Meloidogyne hapla. Attachment of microbes to J2 in these three soils was investigated by cultivation-independent methods to identify those which specifically interacted with J2 in the most suppressive soil (Kleinwanzleben). The three soils differed in the microbes attached to J2. PCR-DGGE fingerprints of amplified ITS fragments or 16S rRNA genes showed many fungi and bacteria that were abundant on J2 but not in the surrounding soil, some of which seemed to be present in all three soils while most were soil type specific. Many bacteria associated with J2 from the most suppressive soil were closely related to infectious species like Shigella spp., while most abundant were Malikia spinosa and Rothia amarae as determined by 16S rRNA gene amplicon pyrosequencing. Nematode-fungus disease complexes can cause dramatic synergistic yield losses. Bacterial strains known as antagonists of phytopathogenic fungi were evaluated with respect to their biocontrol potential towards M. incognita. Seed inoculation with most of the strains significantly reduced propagation of nematode on tomato roots. The best strains Bacillus subtilis Sb4-23, Mc2-Re2, and Mc5- Re2 were further studied for their mode-of-action. The strains were able to affect the nematodes directly by metabolites present in culture supernatants and indirectly by induced systemic resistance of the plant. Experimental comparison of direct and plantmediated effects suggested that the latter was the major control mechanism of these antagonists. Overall, these findings may improve the basis for integrated management strategies of root-knot nematodes in organic farming

Crown Gall Disease in Moroccan Almond Trees: Tumorigenic Bacteria and Sustainable Management through Biological Control

Crown gall is a globally recognized bacterial disease caused by Agrobacterium tumefaciens. This bacterium is characterized by its potential to infect a wide range of plants, specifically fruit trees. In Morocco, almond trees (Prunus amygdalus) are among the most heavily affected species by this disease. In our study, all tested bacteria were isolated in the laboratory from infected almond samples exhibiting tumors at the crown and root of the sampled trees collected from northwestern of Morocco. Biochemical, pathogenicity and molecular identification were performed to identify the causal agent of almond crown gall. Molecular identification was done using a duplex PCR (A/C9 and A/E9) targeting virD2gene located in the pTi plasmid, which is the oncogenic element of the bacterium. The results showed that 12 from 20 isolates were identified as tumorigenic A. tumefaciens with the presence of pTi. Moreover, these isolates were found to exhibit tumorigenic properties both in vitro and in vivo when tested on indicator plants, specifically tomato and carrot discs. The efficacy of agrocin synthesized by Agrobacterium radiobacter strain K1026, a biopesticide known as NoGall, was tested in vitro and in planta against tumorigenic strains of A. tumefaciens. Our results revealed a significant inhibitory effect in vitro, with inhibition percentages ranging from 29.5% to 38.9%. Additionally, we observed a complete reduction in tumor growth at the inoculation sites on indicator plants, with a 100% reduction percentage

Perspective of beneficial microbes in agriculture under changing climatic scenario: a review

Agriculture is a complex network of interactions of plants with microorganisms. There is a growing demand for ecologically compatible environment friendly technique in agriculture that might be able to provide adequate supply of nutrients for the increasing human populations through improvement of the quality and quantity of agricultural products. Under the changing climatic scenario of global fluxes of the key biogenic greenhouse gases (CO2, methane and nitrous oxide), and some other environmental problems, the application of beneficial microorganisms in agriculture would serve as an important alternative gateway to some of the traditional agricultural techniques. Microorganisms of agricultural importance represent key ecological strategy for integrated management practices like nutrient management, disease and pest management in order to reduce the use of chemicals in agriculture as well to improve cultivar performance. The present review is intended to focus on the emergence of agriculturally important microorganisms (AIMs) to develop an ideal agricultural system through efficient utilization of nutrients and recycling of energy and thereby to preserve the natural ecosystem resources under climate change. The progress to date in using the beneficial microflora in a variety of applications related to agriculture along with key mechanism of action is also discussed in this review

The 1-aminocyclopropane-1-carboxylic acid deaminase-producing Streptomyces violaceoruber UAE1 can provide protection from sudden decline syndrome on date palm

In the United Arab Emirates (UAE), sudden decline syndrome (SDS) is one of the major fungal diseases caused by Fusarium solani affecting date palm plantations. To minimize the impact of the causal agent of SDS on date palm, native actinobacterial strains isolated from rhizosphere soils of healthy date palm plants were characterized according to their antifungal activities against F. solani DSM 106836 (Fs). Based on their in vitro abilities, two promising biocontrol agents (BCAs), namely Streptomyces tendae UAE1 (St) andStreptomyces violaceoruber UAE1 (Sv), were selected for the production of antifungal compounds and cell wall degrading enzymes (CWDEs), albeit their variations in synthesizing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD). Although both isolates showed antagonism when applied 7 days before the pathogen in the greenhouse experiments, the ACCD-producing Sv was relatively superior in its efficacy against SDS over the non-ACCD-producing St. This was evident from the symptoms of SDS in diseased date palm seedlings which were greatly reduced by Sv compared to St. On a scale of 5.0, the estimated disease severity indices in Fs-diseased seedlings were significantly (P < 0.05) reduced from 4.8 to 1.5 and 0.5 by St and Sv, respectively. Thus, the number of conidia of Fs recovered from plants pre-treated with both BCAs was comparable, but significantly (P < 0.05) reduced compared to plants without any BCA treatment. In addition, a significant (P < 0.05) decrease in ACC levels of both the root and shoot tissues was detected inSv + Fs seedlings to almost similar levels of healthy seedlings. However, in planta ACC levels highly increased in seedlings grown in soils infested with the pathogen alone or amended with St prior to F. solani infestation (St + Fs). This suggests a major role of ACCD production in relieving the stress of date palm seedlings infected with F. solani, thus supporting the integrated preventive disease management programs against this pathogen. This is the first report of effective rhizosphere actinobacterial BCAs to provide protection against SDS on date palm, and to help increase agricultural productivity in a more sustainable manner in the UAE and the other arid regions

Seeing the forest for the trees : Use of phages to treat bacterial tree diseases

Trees and woody plants can be attacked by many pests and pathogens either individually or as polymicrobial infections. In particular, infections caused by tree-specific bacterial pathogens have become more common during the last decade, causing serious concern for important tree and woody plant species in horticulture, urban environments, and forests. For example, Xylella and Pseudomonas bacteria are causing significant economic and ecological devastation throughout Europe in olive, cherry, and other stone fruits, mainly because of lack of efficient control methods and the emergence of bacterial resistance to traditional antimicrobial compounds such as copper and antibiotics. Hence, there is an urgent need for innovative approaches to tackle bacterial plant diseases. One way to achieve this could be through the application of biological control, which offers a more environmentally friendly and targeted approach for pathogen management. This review will explore recent advances in use of pathogen-specific viruses, bacteriophages (or phages), for the biocontrol of bacterial tree diseases. Phages are an important component of plant microbiomes and are increasingly studied in plant pathogen control due to their highly specific host ranges and ability to selectively kill only the target pathogenic bacteria. However, their use still poses several challenges and limitations, especially in terms of managing the bacterial diseases of long-lived trees. A particular insight will be given into phage research focusing on controlling Pseudomonas syringae pathovars, Erwinia amylovora, Xanthomonas species, Ralstonia solanacearum, and Agrobacterium tumefaciens. Recent milestones, current challenges, and future avenues for phage therapy in the management of tree diseases are discussed

Heterobasidion bref. and armillaria (fr.) staude pathosystems in the basque country: identification, ecology and control

114 p.In the temperate forest, root and butt rot fungi are considered the greatest causes of economic losses. Armillaria and Heterobasidion species are often the causative agents of this disease which is characterized by chlorotic leaves, progressive thinning of the crown, slower leader growth, and rapid tree death. In the present study, the distribution of Armillaria and Heterobasidion in the Basque Country, and the environmental factors associated with both fungal complexes, were described. The species and population diversity of both genera in selected plantations and native forests were determined, and host range in the field and host susceptibility to A. mellea under greenhouse conditions were established. The results contribute to a better understanding of the epidemiology of these forest pathogens. In addition, bacteria native to the P. radiata rhizosphere that are able to reduce pathogenic effects of A. mellea and H. annosum s.s in young P. radiata trees were isolated and characterized. The compiled information will facilitate the development of management strategies, especially in areas of the Basque Country where the problem of replanting forests infested by diverse native and exotic pathogens is endemic.Neiker-Tecnalia. Eusko Jaurlaritza. Gobierno Vasco FINBIF Innovation INIA : Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria Healthy Fores

Heterobasidion bref. and armillaria (fr.) staude pathosystems in the basque country: identification, ecology and control

114 p.In the temperate forest, root and butt rot fungi are considered the greatest causes of economic losses. Armillaria and Heterobasidion species are often the causative agents of this disease which is characterized by chlorotic leaves, progressive thinning of the crown, slower leader growth, and rapid tree death. In the present study, the distribution of Armillaria and Heterobasidion in the Basque Country, and the environmental factors associated with both fungal complexes, were described. The species and population diversity of both genera in selected plantations and native forests were determined, and host range in the field and host susceptibility to A. mellea under greenhouse conditions were established. The results contribute to a better understanding of the epidemiology of these forest pathogens. In addition, bacteria native to the P. radiata rhizosphere that are able to reduce pathogenic effects of A. mellea and H. annosum s.s in young P. radiata trees were isolated and characterized. The compiled information will facilitate the development of management strategies, especially in areas of the Basque Country where the problem of replanting forests infested by diverse native and exotic pathogens is endemic.Neiker-Tecnalia. Eusko Jaurlaritza. Gobierno Vasco FINBIF Innovation INIA : Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria Healthy Fores

Inventory and world geographical distribution of the olive tree (Olea europaea L.) diseases caused by viruses, bacteria and phytoplasma

The objective of this work is to prepare an Inventory about the viral and bacterial diseases of the olive tree (Olea europaea L.) and to study their Geographical distribution in the world. Fifteen viruses were considered as pathogens on the olive tree, these viruses were reported in 22 countries in five continents showing different symptoms on different olive tree varieties. All viruses were reported in Italy, some of these viruses were found only in the Eurepean continent, such as Olive vein yellows associated virus (OVYaV), Olive yellow mottling and decline associated virus (OYMDaV), Olive mild mosaic virus (OMMV) and Olive semilatent virus (OSLV). Two bacteria were reported as pathogen on the olive tree; Pseudomonas savastanoi. pv. savastanoi responsible bacteria of thr olive knot; a serious disease of olive (Olea europaea L.) in the Mediterranean region. This bacteria was detected in 22 countries in five continents, the majority of these countries exist in the Mediterranean Basin. Another bacteria was Xylella fastidiosa responsible of the quick decline syndrome of olive (OQDS), was deteted in Europe in four countries (Italy, Spain, Portugal and Slovenia) and in America in three countries (USA, Argentina and Brazil). Phtoplasmas was reported in 5 countries in three continents (Europe, Asia and Australia)