Academic literature on the topic 'Phytopatogenic microorganisms'

Aim. Phytopathogenic microorganisms are one of the main causes of agricultural productivity losses. Thereby, the goal of this study was to evaluate actinomycetes strains, isolated from Juniperus excelsa Bield. rhizosphere, antagonistic activity against plant pathogenic bacteria and fungi. Methods. In this study we used microbiological methods for isolation actinomycetes from rhizosphere. Antagonistic activity was evaluated by using the dual culture method. Results. 372 actinomycete stains were isolated from J. excelsa Bield. rhizosphere. More than 60 % actinomyces isolates showed antibacterial activity against to lest one of the tested phytopathogenic bacteria genus Pseudomonas, Pectobacterium, Agrobacterium, Erwinia, Xanthomonas and 20.5 % of the tested phytopathogenic fungi genus Aspergillus, Alternaria, Fusarium, Botrytis. Only 2 strains had antagonistic activity to the all of the tested microorganisms and 62 strains, which had antagonistic activity to the one test-microorganism. Conclusions. Actinomicetes of J. excelsa Bield. rhizosphere are source for bioactive compounds against phytopatogenic microorganisms and showed good biotechnology potential. These results are the first step to the screening new biopesticides for controlling phytopatogenic diseases in plan. Keywords: actynomicetes, phytopathogens, biocontrol.

Objective: The purpose of this literature review is to know the different phytopathogenic fungi present in these two types of environments, and the factors involved in their presence or absence. Materials and methods: For the construction of this article, a bibliographic search was carried out in the following databases: Pubmed, Science direct, Google scholar, Scielo and NCBI in English and Spanish, using the following descriptors: Fungi, Aquatic microorganisms, and Fungi in soil. Results: In this bibliographic review, the presence of Aspergillus sp, Fusarium sp, Mucor sp, Penicillium sp, Alternaria sp, Trichoderma sp, Blastomyces sp, Geotrichum sp, etc. was found in most of the aquatic environments. In addition, in terrestrial environments, fungi of the genus Rhizopus sp, Fusarium sp, Beauveria sp, Absidia sp, Mucor sp, Aspergillus sp, Penicillium sp, Paecilomyces sp, etc. were found. Likewise, we investigated the factors that determine the concentration and diversity in aquatic and terrestrial environments, in all types of fungi. In aquatic environments, these are: temperature, rainfall, water velocity, nutrient status, anthropogenic impact and abundance of decomposing matter. On the other hand, in terrestrial environments, we find: pH, anthropogenic impact, and the presence of pollutants. Conclusion: In the case of fungi in fresh water, the genus Aspergillus sp was the most representative, while in the terrestrial environment the genus Fusarium sp, and Rhizopus sp, were the most predominant with their appearance in most of the studies cited in this review.

Antibiotic production by plant-associated microorganisms represents an environmentally compatible method of disease control in agriculture. However, a vide application of bacterial strains needs careful selection and genetic characterization. In this investigation, selected Pseudomonas strains were characterized by rep-PCR methods using ERIC and (GTG)5 primers, and partial 16S rDNA sequence analysis. None of strains produced homoserine lactones (C4, C6, C8) as quorum sensing signal molecules. Very poor production of phenazines and no significant fungal inhibition was observed for PS4 and PS6 strains. High amount of phenazines were produced by Pseudomonas sp. strain PS2, which inhibited mycelial growth of 10 phytopatogenic fungi in percent of 25 (Verticillium sp.) to 65 (Fusarium equiseti). Genetic characterization of the Pseudomonas sp. PS2 and evaluation of phenazines production, as the main trait for growth inhibition of phytopathogenic fungi, will allow its application as a biosafe PGPR for field experiments of plant disease control.

Abstract Antagonistic activity of microorganisms against phytopathogens is mainly the results of plants’ health improvement due to the inhibition of pathogens growth and the induction of plants resistance against diseases. The aim of the research was to determine antagonistic properties of Pantoea agglomerans against Rhizoctonia solani. The properties of two strains P. agglomerans BC17 and BC45 were assessed according to the following criteria: mycelial growth of R. solani in the presence of bacterial metabolites, an impact of P. agglomerans on the growth of sugar beet in the pots containing soil with and addition of R. solani and without it, the ability to produce indole-3-acetic acid (IAA). It has been recorded that antagonistic properties of tested strains are different. In the presence of metabolites of BC17 strains, the mycelial growth of R. solani was inhibited by 78 % and for the strain BC45 the value amounted 46 %. In the pot bioassay the number of infested plants growing in the soil inoculated with P. agglomerans and the pathogen was lower when compared with the pots containing R solani. A higher reduction of infested plants, amounting 23 %, was obtained for the strain BC17. Both strains had the ability to produce IAA - a plant hormone of the auxin class, in the presence of tryptophan and its absence in the medium. The highest concentration of IAA was recorded after 7 days of culturing in the supernatant obtained from the media containing 2000 μg/cm3 of tryptophan. For the strain BC17 the concentration of IAA marked in the post - culturing liquid amounted 71.57 μg/cm3, and for the BC45 strain it amounted over 80 μg/cm3. Obtained results prove that P. agglomerans may be used in the biological protection against phytopatogenic strains of R. solani.

Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field. Factors limiting mycoherbicide efficiency include temperature and humidity. Results from this thesis indicate that solar radiation has both a damaging effect(reduction in germination)limiting efficacy and a photomorphogenic effect(appressorium induction)increasing efficacy. The study has also shown significant interaction between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as third major component of the environment that should be considered when trying to produce mycoherbistats. With the findings presented in this thesis and further research on disease development under different conditions, in combination with the formulation of conidia in suitable UV protectants, a computer simulation modelling the conditions leading to epidemics caused by C.orbiculare, D.avenacea and R.alismatis could be constructed. It may be possible to manipulate fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed.
Doctor of Philosophy (PhD)

The plant pathogen Ralstonia solanacearum is the causal agent of the devastating disease known as bacterial wilt. This disease affects more than 200 plant species in over 50 families, including economically important crops such as tomato, potato, banana or pepper. R. solanacearum is a soil-borne and vascular pathogen. It enters its hosts through wounds in the roots and lateral root emerging sites. Then, the pathogen traverses the cortical apoplast until it reaches the vascular cylinder and colonizes to very high numbers the xylem vessels. The combination of bacterial growth and the secretion of a mucus-like exopolysaccharide clogs the infected vessels, blocking its water flow and causing the wilt disease symptoms that lead to the dead of the plant. To date, the most reliable management strategy to control R. solanacearum has been the use of genetic resistance. In this thesis we attempted to shed light on the old question of bacterial wilt resistance, which has haunted plant epidemiologists for more than one hundred and twenty years, using novel proteomic techniques. We thought that the best way to address this challenge was to follow a “divide and conquer” approach: by identifying the key bottlenecks of resistance (Chapter 1), we would be able to precisely dissect which proteins are involved in different tissues and stages of the infection (Chapters 2 and 3). Finally, in Chapter 4 we delved into the tomato P69 family of subtilisin-like proteases, as many of its members appeared upregulated during infection. In Chapter 1 we identified the plant tissues and organs that represented major constraints during R. solanacearum colonization. Using simple and very visual tools –bacterial reporter strains and plant grafting–, we highlighted the root system as the first and most conspicuous barrier. The roots consist of an epidermis and a cortical parenchyma that R. solanacearum must traverse before reaching the vasculature, a movement that occurs through the intercellular space or apoplast. In this chapter, we showed how the roots of resistant plants (H7996) effectively prevented R. solanacearum entry and invasion, single-handedly reducing the colonization and consequent development of wilt disease symptoms in grafted plants. A reduction in colonization was also evident within the xylem vessels of H7996. Grafting susceptible roots onto resistant hypocotyls allowed us to confront the resistant stems with high bacterial inoculum using the conventional –and more natural– soil-drench inoculation method. Once the bacteria faced the resistant tissue, a decrease in colonization was observed, indicating that H7996 can also restrict R. solanacearum movement/growth along the xylem in a root-independent manner. In addition, we observed how this resistant tomato variety impeded the escape of R. solanacearum from colonized xylem vessels towards the apoplastic space of the neighboring parenchymatic cells, a situation clearly promoted in susceptible plants (Marmande) and that facilitated tissue decay in the latest stages of infection. Altogether, the data presented in this chapter highlighted these two plant environments, the apoplast and the xylem, as the two main battlefields of infection. In Chapters 2 and 3 we used quantitative mass spectrometry approaches to investigate the apoplastic and xylem sap proteomes in response to R. solanacearum. In Chapter 2 we focused on the active proteome of the apoplast using activity-based molecular probes that covalently bound to the active site of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs). Upon infection, the activity of PLCPs and SHs was higher in the apoplast of H7996 than in Marmande. We found that two well-known PLCPs, Rcr3 and Pip1, and many SHs from distinct protease families experienced significant changes after R. solanacearum inoculation and in the comparison of the two tomato varieties. Additionally, a protein network analysis suggested that the apoplastic proteome of H7996 might be more naturally prepared to face intruders, as many protein interactions did not substantially change upon infection. In Chapter 3 we focused on the xylem sap proteome and, similar to the apoplast, we observed many differentially accumulated proteins (DAPs) in response to infection. A clear “downregulation” was detected in Marmande, which could either be due to a deliberated inhibition caused by R. solanacearum or to tissue damage resulting from the infection process. On the other hand, cell wall modifying enzymes, including peroxidases and glycoside hydrolases (GHs), were overaccumulated in H7996. Moreover, the overaccumulation of many tomato and R. solanacearum GHs both in the apoplast and the xylem highlighted the importance of the cell wall as an effective barrier in plant-pathogen interactions, particularly against R. solanacearum. Finally, in the survey of both proteomes –apoplast and xylem–, we identified members of the P69 family of subtilases to overaccumulate upon R. solanacearum infection. This 10 gene family of proteases was comprehensibly addressed in Chapter 4. We analyzed the conservation of this family and found that they are only present in Solanaceae, being P69A and P69D the two most conserved P69 genes. P69s were not only induced by R. solanacearum but responded to unrelated plant pathogens, as illustrated by several transcriptomic and proteomic studies. Additionally, their overall high percentage of identity (>71%) appeared to partially translate in their cleavage specificity, although some particularities could be drawn for P69A, P69B and P69D. Conversely, substrate specificity might also be determined by their differential post-translational N-glycosylation status, and/or by the higher sequence divergence within their protease- associated domain, which could influence protein-protein interactions. Finally, a CRISPR/Cas9 mutagenic approach was conducted to obtain a P69D single mutant and two separate P69-cluster deletion mutants. We assayed the susceptibility of the P69D single mutant and found a slight yet significant increase in susceptibility, probably caused by an increased growth of the pathogen in its root system. The potential role of P69D in defense and other aspects regarding P69 subtilases are addressed.
El fitopatogen Ralstonia solanacearum és el bacteri causant de la devastadora malaltia coneguda com a marciment bacterià. Aquesta malaltia afecta més de 200 espècies de plantes de 50 famílies diferents, incloent importants plantes de cultiu com la tomaquera o la patatera. R. solanacearum entra als seus hostes a través de petites ferides en les arrels o en els punts en que emergeixen les arrels laterals. Un cop ha penetrat l’arrel, R. solanacearum es mou seguint una via apoplastica –a través dels espais intercel·lulars– fins que arriba als feixos vasculars de la planta, on colonitza el xilema. En el xilema, aquest bacteri es multiplica en grans quantitats i comença a secretar una substància mucosa que acaba col·lapsant el feix vascular. La falta d’un flux hidràulic continu provoca l’aparició dels clàssics símptomes de marciment, que desemboquen en la mort de la planta. Ara per ara, la millor estratègia per controlar R. solanacearum és l’ús de varietats de cultius resistents. En aquesta Tesi Doctoral hem utilitzat noves tècniques d’anàlisi de proteïnes per caracteritzar la resposta de les plantes resistents a R. solanacearum, utilitzant la planta de la tomaquera com a model. Primerament hem caracteritzat quins són els principals colls d’ampolla que restringeixen la colonització del patogen en tomaqueres resistents, identificant els espais intercel·lulars –o apoplast– i el xilema com els camps de batalla més importants entre la planta i R. solanacearum (Capítol 1). Posteriorment (Capítols 2 i 3), hem analitzat el context proteic d’aquests dos ambients en resposta al patogen comparant una varietat susceptible (Marmande) amb una de resistent (H7996). Finalment, en el Capítol 4, hem aprofundit en la caracterització d’una família de proteases concreta, conegudes com a P69, ja que certs membres de la família s’acumulaven tant en l’apoplast com el xilema en resposta a R. solanacearum. En el Capítol 1 hem identificat quins teixits i òrgans restringeixen el moviment de R. solanacearum dins la planta. Utilitzant soques reporteres del bacteri –emissores de llum o fluorescents– i la tècnica de l’empelt, hem observat com l’arrel és la primera i més important barrera contra el patogen. Així, les arrels de plantes resistents eren capaces de limitar l’entrada del patogen a la planta o bé reduir-ne la multiplicació. Empeltant arrels susceptibles en tiges resistents, hem aconseguit infectar les tiges de plantes resistents amb un inòcul bacterià molt més gran del normal –similar al que es troben les plantes susceptibles–. En fer-ho hem observat una disminució en la colonització dins dels vasos xilemàtics de la part resistent, indicant que en la tija actuarien uns mecanismes de defensa independents de l’arrel. A més, hem pogut observar com, la varietat resistent impedeix que el bacteri s’escapi dels feixos vasculars infectats. Aquest fet no es dona en plantes susceptibles, on el bacteri envaeix els espais intercel·lulars de les cèl·lules parenquimàtiques circumdants. Finalment, hem definit un llindar de massa bacteriana en l’hipocòtil per sobre de la qual les plantes es marceixen. Un cop identificats l’apoplast i el xilema com a principals camps de batalla entre la planta i R. solanacearum, en els Capítols 2 i 3 hem caracteritzat la resposta proteica de la planta en aquests dos compartiments. En el Capítol 2 s’ha investigat l’activitat de cisteïna i serina proteases (PLCP i SH, respectivament, de les seves sigles en anglès) de l’apoplast. En resposta a la infecció, la varietat resistent mostra un increment en l’activitat d’aquestes famílies de proteases. Mitjançant espectrometria de masses hem identificat canvis en l’acumulació de 4 PLCPs i 27 SHs en les dues varietats davant la infecció. Entre les PLCPs, hem identificat les ja caracteritzades Rcr3 i Pip1. A més, moltes SHs encara no han estat caracteritzades. Addicionalment, amb la construcció d’una xarxa d’interaccions proteiques hem pogut denotar com la varietat resistent, al contrari de la susceptible, experimenta menys canvis topològics davant la infecció. Aquests fets indicarien que aquesta varietat està més preparada per respondre al patogen en el seu estat basal. En el Capítol 3 ens hem endinsat en l’estudi proteòmica del xilema. Com en l’apoplast, també s’ha observat que moltes proteïnes s’acumulen de forma diferent en resposta a la infecció. És més, s’ha observat una clara disminució de proteïnes secretades en les plantes susceptibles, que pot ser deguda a una inhibició deliberada de la resposta immunològica per part de R. solanacearum, o bé al mateix dany tissular causat pel seu creixement. Per altra banda, davant la infecció s’observa una clara acumulació xilemàtica de peroxidases, glicosil hidrolases i proteases, cosa que reforça la importància de l’enfortiment dels vasos i les parets cel·lulars com un dels mecanismes claus per combatre la infecció. Finalment, tant en l’apoplast com el xilema hi hem identificant membres de la família de subtilases coneguda com a P69. Aquestes proteases s’acumulen en els dos compartiments davant la infecció. La seva caracterització es presenta al Capítol 4. Es tracta d’una família de 10 gens (en tomaquera) altament conservats, ja que les seqüències de les proteïnes comparteixen més d’un 71% d’identitat. S’ha constatat que les P69s són específiques de solanàcies, tot i que no totes es troben conservades en les espècies que hem analitzat (patatera, alberginiera, pebrotera i tabac). No només s’acumulen per la infecció de R. solanacearum, sinó que altres patògens també en provoquen la inducció. A més, analitzant l’especificitat de tall, s’ha observat que poden tallar seqüències similars, tot i que P69A, P69B, P69D presenten certes especificitats. Un anàlisis in silico ha determinat que aquestes proteïnes pateixen diferents modificacions post-traduccionals en forma de N-glicosilació, cosa que podria afectar la seva especificat de substrat en la mateixa planta. Finalment, s’ha utilitzat la tecnologia CRISPR/Cas9 per crear delecions en un gen concret (P69D, ja que només es va detectar actiu en plantes resistents), o bé eliminar per deleció el màxim nombre de gens de la família. S’ha caracteritzat una lleugera susceptibilitat a R. solanacearum per part del mutant per P69D, que probablement s’atribueix a una facilitat per part del patogen de colonitzar el sistema radicular.

Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field. Factors limiting mycoherbicide efficiency include temperature and humidity. Results from this thesis indicate that solar radiation has both a damaging effect(reduction in germination)limiting efficacy and a photomorphogenic effect(appressorium induction)increasing efficacy. The study has also shown significant interaction between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as third major component of the environment that should be considered when trying to produce mycoherbistats. With the findings presented in this thesis and further research on disease development under different conditions, in combination with the formulation of conidia in suitable UV protectants, a computer simulation modelling the conditions leading to epidemics caused by C.orbiculare, D.avenacea and R.alismatis could be constructed. It may be possible to manipulate fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed.