Microbial species used for the biological control of phytopathogens

Liliana Bárcena, Alba Domingo, Bruna Hernández i Estel Vallmajó. 

Microbes as biocontrol agents refers to the use of biological methods to control plant diseases and pests instead of insecticides or pesticides which are toxic and harmful to humans and animals as they pollute crops, vegetables, fruits and also the soil and groundwater. In conventional farming the use of chemicals is extended but there is an increasing need of understanding the flora and fauna’s interactions so that we can eradicate those chemicals. 

Biocontrol measures allow to reduce dependence on chemicals by becoming more familiar with various life forms that inhabit land (pest and predator), life cycles, patterns of feeding and the habitats that they prefer in order to develop appropriate means of biocontrol.

In this blog we are going to discuss the biological control of phytopathogens.

A phytopathogen is a microorganism that generates diseases in plants, through disturbances in cellular metabolism, by secreting enzymes, toxins and other substances. They can be bacteria, viruses, protozoa, fungi, nematodes and molluscs. (Agrios, 1997)

Plant diseases induced by plant pathogens are the most difficult to control. It’s interesting to treat plant diseases by biological control with micro-organisms. The most studied groups of biological control agents are Pseudomonas spp (genus of gram negative bacteria) and Trichoderma spp (genus of fungi). The success of biological control depends on plant-microbial interactions and on the ecological fitness of the biological control agents. (Alabouvette et al., 2009).

Image 1. Pseudomonas aeruginosa on an agar plate. (Cornelis, 2008)

 

Methods of infection:

The main methods of infection by phytopathogens are through destroyer enzymes of the cellular wall, toxins and bacterial effector proteins.

-       Destroyer enzymes of the cellular wall degrade the vegetal cellular wall to free the inner nutrients.

-       Toxins: can affect all plants or can only cause damages to one host plant.

-       Bacterial effector proteins are injected by bacterial cells into the cells of their host. This injection is mediated by type 3 secretion systems (TTSS or T3SS) which is a protein appendage found in several gram-negative bacteria.

Mechanisms of biological control agents:

Biocontrol activities of microorganisms include the production of antibiotics, lytic enzymes and induction of systemic resistance in the host plant (Image 2). 

Image 2. Mechanisms of biocontrol for phytopathogens.

Microbial antagonism results from the interactions between two different micro-organisms that share the same ecological niche. There are different types, like parasitism, competition for nutrients and antibiosis.

-       The parasitism of a plant pathogen by other microorganisms is an extended distributed phenomenon. The parasitic activity of strains of Trichoderma spp (Image 3) towards pathogens has been extensively studied (Chet & Baker, 1981). It involves specific recognition between the antagonist and its target pathogen and several types of cell wall-degrading enzymes that enable the parasite to penetrate the hyphae of the pathogen. This type of antagonism, which causes death of the target organism, mainly results in a decrease in the inoculum density. Parasitism of fungal pathogens by viruses or virus-like particles can also induce hypovirulence. Under favorable conditions, hypovirulence can spread naturally in diseased forests (Milgroom & Cortesi, 2004).

Image 3. Trichoderma attacking a plant pathogen (Rhizoctonia sp, cause of root rot). From: («Trichoderma Attacking A Plant Pathogen A Narrow Hyphae Of Trichoderma Coil Around Wide Hypha Of Rhizoctonia The Latter Will Collapse And Die Trichoderma Is A Biological Control Agent Sem Magnification 2350x Foto de stock | Getty Images», s.d.)

   In hyperparasitism, the pathogen is directly attacked by a specific biological control agent that kills it or its propagules. In general, there are four major classes of hyperparasites: obligate bacterial pathogens, hypoviruses, facultative parasites, and predators.

-       Predation is any bacteria that kill other microbes and consume them as a nutritional resource.

-       The competition for nutrients regulates the population dynamics of microorganisms with the same ecological niche and with similar physiological requirements when resources are limited. This competition is usually caused by carbon in the soil buy it can also be because of other elements such as iron.

-       The antibiosis is the biological interaction which consists in the impossibility of the coexistence between some organisms with others that produce some substances that causes them death. These substances can be antibiotics sensu stricto, bacteriocins, enzymes such as CWDE (cell wall-degrading enzyme) and volatile compounds with antifungal activity.

Induced resistance of the plant means that plants have active defense apparatuses that can be actively expressed in response to biotic stresses (pathogens and parasites) of various scales (ranging from microscopic viruses to phytophagous insect). If defense mechanisms are triggered by a stimulus prior to infection by a plant pathogen, disease can be reduced. Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated. Systemic acquired resistance and induced systemic resistance are two forms of induced resistance where in plant defenses are preconditioned by prior infection or treatment that results in resistance against subsequent challenge by a pathogen or parasite.

Lytic enzymes and other byproducts of microbial life. Many microorganisms produce lytic enzymes that can hydrolyze variety of polymeric compounds, including chitin, proteins, cellulose, hemicellulose, and DNA. Expression and secretion of these enzymes by different microbes can sometimes result in the inhibition of plant pathogen activities directly. Microbes that show a preference for colonizing and lysing plant pathogens might be classified as biocontrol agents. 

Image 4. Trichoderma harzianum attacking the phytopathogen Pythium ultimum (green). («Trichoderma Control de Hongos Fitopatógenos | Intagri S.C.», s.d.)

 

Pythium ultimum is a plant pathogen that causes the killing or weakening of the seeds or seedlings before or after they germinate. It affects different plants like corn, soybean, potato, fir and many other ornamental species. Other fungus used as biological control agents against Pythium ultimum are Candida oleophila, Gliocladium catenulatum and Trichoderma virens and it also includes the bacteria Bacillus subtilis and Streptomyces griseoviridis (Farr and Rossman, 2014) Table 1. Species of the genus Trichoderma used in the biological control of fungi and phytopathogenic bacteria in different crops   Species Antagonist against Crop and who reports it T. atroviride Rhizoctonia solani Botrytis cinerea Sclerotinia sclerotium Armillaria mellea Potato (Lahlali and Hijri, 2010) Strawberry (Fraize et al., 2003) Alfalfa (Savazzini et al., 2008) Grape (Savazzini et al., 2008) T. asperellum Fusarium oxysporum Pseudomonas sywingae Pythium sp. Tomato (Cotxarrera et al., 2002; Segarra et al., 2010) Cucumber (Trillas et al., 2006) Tomato (Aerts et al., 2002) T. hamatum Fusarium oxysporum Sclerotinia minor Botrytis cinerea Xanthomonas vesicaria Radish (Heremans et al., 2005) Lettuce (Rabeendran et al., 2006) Begonia (Horst et al., 2005) Tomato (Alfano et al., 2007) T. harzianum Botrys cinerea Sclerotinia sclerotium Pseuperonospora cubensis Sphaeroteca fusca Fusarium oxysporum Rhizoctonia solani Pythium sp. Phaeomoniella chlamydospora Cucumber (Elad, 2000), tomato (Fiume, 2006) Cucumber (Elad, 2000) Cucumber (Elad, 2000) Cucumber (Elad, 2000) Melon (Lopez et al., 2010), grapevines (El-mohamedy ket al. 2008) Tomato (Strashnov et al., 1985; Amer and Abou-El-Seoud, 2008). radish (Lee et al., 2008) Corn (Herman et al., 2004) Grapevines (Di marco and Osti, 2007) -       Biological control of the fireblight. The fireblight is a disease caused by bacteria from the specie Erwinia amylovora and affects mainly rosaceae family plants which include fruit trees and ornamental plants with high economic and gastronomic value. The species used for biological control are competing bacteria of Erwinia amylovora like: Pantoea agglomerans, Pseudomonas fluorescens and Bacillus subtilis. Even though these bacteria are only effective when they’re applied during the process of blooming of the plants, because the main mechanism of performance is to avoid the infection of the flowers by Erwinia amylovora (whose infection is basically found in the flowers, so that if those are damaged the fruit production will be drastically reduced). However this method does not replace the chemical treatment, but only reduces the application dose. This problem was affecting different Spanish regions so that the government included it in the RD 1201/1999 that establish the national program of eradication and control on fireblight on rosaceae. («BOE.es - Documento consolidado BOE-A-1999-16747», s.d.).   Image 5. Erwinia amylovora or fireblight on pear trees. («Erwinia amylovora - Wikipedia, la enciclopedia libre», s.d.). -       Biological control on powdery mildew caused by the fungus. This fungus attacks the leaves and the young shoots of different plants but especially the vineyard. In this case the especies used is a fungus from the Ascomycetes group, Ampelomyces quisqualis. The most used method to inoculate the fungus consist of the elaboration of products whose active ingredient is formed by the spores of the mycoparasitic fungi. This product is applied to the damaged plants, preferently at the first hours of the afternoon as the spores of the fungus require a high relative humidity to start the process and it is by the early afternoon that the humidity rises. Once the spores of A. quisqualis have been settled on the parasit and have managed to germinate, the fungus starts to develop inside the parasite’s hyphens and the growth of the reproductive structures weakens the parasit fungus of the powdery mildew and if the environmental conditions allow this process to repeat the growth of the parasit will be reduced within the next few days.  Image 6. Powdery mildew. (Pérez-García et al., 2009) There is an alternative environmentally friendly for controlling phytopathogen, without using fungicides, focoused on microorganisms producing mycolytic enzymes, especially chitinases, which are known to hidrolyze chitin, a major component of fungal cell walls. Chitinolytic fungi comprise 25-60% of the total mold fungi, but their number is lower than the number of bacteria. Chitinolytic microorganisms for a potential biotechnological application may be produced in various natural environments. Their application is not limited to degradation of the waste containing chitin. Numerous studies demonstrated the possibility of using them in production of chitinolytic phytopathogens. In biological control of fungal phytopathogens, application of agents containing various metabolites of microorganisms, including chitinases, appears to be more efficient and seems to bring better results in fighting fungal phytopathogens. The chitinolytic microorganism actinomycetes are the most important.   Table 2. Biochemical properties of several microbial chitinases. (PDF Chitinolytic Microorganisms and Their Possible Application in Environmental Protection). Genetically modified biocontrol agents: with genetic engineering techniques it’s also possible to use the recombined enzymes obtained by introducing proper genes encoding antifungal activity into different types of organisms.  Serratia marcescens strain B2 controls fungal diseases on rice effectively. The activity of this microorganism is often negatively affected by abiotic and biotic factors. In the experiment performed by Nobutaka someya and Katsumi akutsu, 2005 disease inhibitory genes were isolated from S. marcescens and put under the control of several types of promoters. These genetically modified microorganisms effectively suppressed rice disease caused by Pyricularia oryzae were not affected by abiotic or biotic factors. Introduction of disease inhibitory genes controlled by promoters and derived from the recipient is a useful technology for the development of biocontrol agents. (Someya & Akutsu, 2005) Another example of genetically modified biocontrol agents is found In the research performed by Bezirganoglu et al. where antifungal protein (AFP) and CHI fusion genes were introduced into oriental melons to control fungal diseases caused by R. solani and F. oxysporum.  Other examples: Pasteuria penetrans Is an obligate bacterial pathogen of root-knot nematodes that has been used as a biological control agent. «Pasteuria penetrans - EcuRed», s.d. Cryphonectria parasitica Is a fungus causing chestnut blight, which causes hypovirulence, a reduction in disease-producing capacity of the pathogen. Muñoz López et al., 2007 Lysobacter and Myxobacteria Are known to produce copious amounts of lytic enzymes, and some isolates have been shown to be effective at suppressing fungal plant pathogens. Kobayashi and El-Barrad 1996, Bull et al. 2002 Pseudomonas chlororaphis It is a bacterium used as soil  inoculant in agriculture and horticulture. Cornelis, 2008   CONCLUSIONS Nowadays the biological control agents are a matter of study and they still need the combination with chemical methods to control phytopathogens and get satisfactory results, in most of the cases they act as a complement to those chemicals treatments and allow to reduce the application dose so that the impact on the environment produced by pesticides, fungicides, etc is lower. Biological control agents can sustain and disseminate by their own, and require little human effort. The animals and plants that are in an area where biological control is used are not usually affected by this method of control. In biological control of fungal phytopathogens, application of agents containing various metabolites of microorganisms, appears to be the more efficient, since they seem to bring better results in fighting fungal phytopathogens. BIBLIOGRAPHY Agrios, G. N. (1997). Plant pathology. Academic Press. 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