Biocontrol of *Phytophthora* Infections

**Chapter 3**

## Plant Beneficial Microbes Controlling Late Blight Pathogen, *Phytophthora infestans*

*Brahim Oubaha, Abdellah Ezzanad and Hernando José Bolívar-Anillo*

#### **Abstract**

Potato (*Solanum tuberosum*) as a food source and culinary ingredient varies is the fourth most produced noncereal crop in the world. Among multiple biotic stresses, late blight caused by *Phytophthora infestans* is the most destructive disease. Control of this pathogen is usually by the synthetic fungicides which have been fueled by the public concern about toxicity and environmental impact and development of pathogens resistance. Biological control agents (BCAs) seems the potentially alternative to these pesticides, biological disease control is now recognized and constitute an important tool in integrated pest management. BCAs strains should be able to protect the host plant from pathogens and fulfill the requirement for strong colonization. Bacteria such as Bacillus, Pseudomonas and Streptomyces and fungi such as Trichoderma and *Penicillium* were the most reported as a BCA against *P. infestans* using different direct antagonistic mode on the pathogen (via e.g. parasitism, antibiosis, or competition) or via exerting their biocontrol activity indirectly by induction in the plant of an induced systemic resistance to the pathogen. In this study, we present an overview and discussion of the use of beneficial microbes (bacteria and fungi) as novel BCAs for biocontrol of *P. infestans*.

**Keywords:** *Solanum tuberosum*, *Phytophthora infestans*, biological control agents, beneficial microbes

#### **1. Introduction**

Plant diseases need a good control strategies in order to maintain the quality and abundance of food around the world. Especially, human population growth has been the source of two major concerns: providing sufficient food for humanity and minimizing worldwide environmental pollution. Several approaches may be used to protect or control plant diseases. Beyond good cultural practices, harvest and postharvest approaches in reduction of pathogen growth, growers often rely heavily on chemical fertilizers and pesticides. However, many countries have reported alarming residues of agricultural chemicals in soil, water, air, agricultural products, and even in human blood and adipose tissue [1, 2]. Additionally, research suggests that the massive use of inorganic fertilizers world-wide is associated with the accumulation of in agricultural soils [3]. Researchers and Policy makers recognize that the excessive and unsystematic application of agrichemical inputs poses a threat to the

environment and humans alike. Consequently, several biologist have focused their efforts on developing alternative inputs to synthetic chemicals for controlling pests and diseases [4]. Among these alternatives those referred to as biological control by using one or more beneficial microbes to suppress the damaging activities of soil-borne pathogens.

Plant growth-promoting microbes (PGPM) are free-living microorganisms of beneficial agricultural importance. The PGPM present important beneficial effects on plant health and growth, suppress disease-causing microbes and improve nutrient. PGPM exist in the rhizosphere and this is defined as the region around the root. PGPM compensate for the reduction in plant growth caused by weed infestation [5], drought stress [6], heavy metals [7], salt stress [8, 9] and some other unfavorable environmental conditions. Beneficial microbes are also the microorganisms that produce hormones, vitamins and growth factors that improve plant growth and increase crop production. Many research reported the ability of this microorganisms to produce indole acetic acid (IAA), gibberellic acid, and cytokinins [10] and production of important metabolites such as siderophores, HCN, and antibiotics that have immense potentiality in enhancing the root surface area, altering root architecture and promoting plant growth. Among the numerous plant growthpromoting microbes (PGPM) are the most commonly applied in the biological control strategies. PGPM may affect plant performance through multiple defense mechanisms against several pathogens, operating directly by the production of specific substances that are able to promote plant growth, increase the availability and uptake of plant nutrients under biotic stress and induce the defense response of plants attacked or indirectly through the suppression of plant pathogen [11, 12].

For the biological control of late blight which is Late blight disease, caused by *Phytophthora infestans* (Mont.) de Bary, is one of the most serious threats to potato production worldwide [13], Applications of different beneficial microbes as a biocontrol bacteria, fungi, algae or their metabolites, have been tested their ability to inhibit potato late blight, and when used as part of an integrated pest management system, they have had varying degrees of success [14–16]. Bacteria with antagonistic activity toward *P. infestans* are found mainly in the genera *Pseudomonas* and *Bacillus*. Although some fungal antagonists such as *Trichoderma atroviride* and *Muscodor albus* showed effective inhibition [17–20]. The objective of this chapter is to review the ability of beneficial microbes used to control late blight of potato caused by *P. infestans*, building on recent detailed reviews and research articles on microorganisms antagonistic to late blight of potato and their management approaches.

#### **2. The methods of isolation of** *P. infestans*

*P. infestans* causes potato and tomato late blight, economically the most important disease of these plant species. The oomycete pathogen is frequently sampled, isolated to pure cultures and stored. Efforts were made to develop isolation and culturing techniques based on tomato and potato. There are two major steps of isolating *P. infestans*, Field collection and isolation of *P. infestans* from infected tissue [21]. Petri dish method makes easier the collection of largest number of diseased samples in the field because is based on selective medium. Leaves, stems, petioles and even slices of diseased tubers can be collected. It allows the transfer of samples from the field to the laboratory in good condition and in turn stimulating sporulation of the lesions for easy isolation. The petri dishes were prepared with 1.5-% water agar, the sample with only one lesion were chosen and placed on the plate lid with the abaxial side up, in such a way so that the agar is on the sample but never in contact. The plates must be

#### *Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*

sealed with Parafilm paper and placed in a cooler. In the laboratory, samples should be incubated at 15–18° C for 3–7 days with light and dark periods of 12 hours and grown hyphal tip of *P. infestans* transferred on a selective medium. Previous reports mentioned the use of some effectiveness selective medium for the isolation of *P. infestans*. The application of fungicides against *P. infestans* can affect the establishement of the oomycetes and their isolation. It is recommended to use tubers from fields where systemic fungicides against *P. infestans* have not been applied. Gamboa et al. [22] reported a method named sandwich method, tuber aseptically were cut in half and quickly place an infected leaflet between both halves. The both halves were attached with adhesive tape and wrap the tuber with paper towels, then place it in a paper or plastic bag for transfer to the laboratory. In the laboratory, the tuber were cuted in slices from the place of contact between the infected leaf and the tuber then put them in a wet chamber and incubate for 7 days to induce pathogen development. Incubation temperature should be 15–18° C with light and dark periods of 12 hours [22]. In the laboratory the isolation *P. infestans* from infected plant tissue can be using different infected tissues from potato or tomato plants. Sporulating lesion on potato/tomato leaves taken from field are washed in fresh water and placed in a humid chamber (inverted petri dish with water agar) with the leaf's abaxial side up and incubated at 15–18°C for 1 day or until fresh sporulation appears. Small pieces of infected tissue from the sporulating border of the lesion are cut and placed under potato/tomato slices in an empty petri dish. Dishes are incubated at 15–18°C for 1 week, until there is abundant sporulation on the upper side of the slice. To re-inoculate leaves, pick sporangia from the top of the tuber and place them in a drop of water on a potato leaf or another tuber slice. If isolating from infected tubers, slice the tuber where infection has occurred and place in a moist chamber until sporulation occurs. When clean inoculum appears on the upper side of an infected tuber slice or leaf, the sporangia are harvested in a flow chamber, by picking them up with an inoculating needle and placing the sporangia on selective medium [23, 24]. Tumwine et al. [25] reported that *P. infestans* grew successfully and well on Rye A agar without the need of antibiotics is one of the recommended medium for the isolation of *P. infestans*. The Rye A agar was described for the first time in 1968 by Caten when Rye B agar were used for the sporulation. However, V8 juice agar (V8A) which is blend of 8 vegetable juices, which supplies the trace ingredients to stimulate the growth of fungi. The acidic pH of the medium favors fungal growth and suppresses bacterial growth. V8A has been one of the most popular and commonly used medium for growth and reproduction of *Phytophthora* species [26]. In 2020, [23] were studied five different media in order to select the optimal culture conditions of *P. infestans*. Modifiations were made to use ingredients available in local markets on the following media: lime bean agar (LBA), Tree tomato or tree tomato agar (TA), carrot agar (AZ), Rye A modifid agar and 32% non-clarifid V8 agar. The findings results showed that as was described before media such as Rye A favored the ability of *P. infestans* to grow effiently.

#### **3. Bacteria**

The use of biological agents to control or suppress *Phytophthora infestans* provides an economic and environmentally friendly approach. As a biopesticides bacteria are the most common and cheaper form of microbial pesticides. The potential of a range of bacterial strains as biocontrol agents of plant pathogens has been reviewed by many scientific reports [27–29]. *Streptomyces*, *Pseudomonas* and *Bacillus* were the most tree bacteria reviewed to control *P. infestans* [30, 31]. *Actinomycetes* were isolated from in general from soil. Samples were diluted to go on serial dilution and plate on humic acid vitamin agar as described by [32] supplemented with an antifungal and antibacterial

Gram- such as nalidixic acid. The isolation plates were incubated at 35 ± 2°C for 7 days. The colonies had been transferred to International Streptomyces Project (ISP) medium No. 2 agar [33, 34] plates for purity check. This isolation method can be improved using same modifications. In the other hand, The isolation methods used to collect *bacillus* and *Pseudomonas* from soil as an endophytic or epiphytic strains were routinely grown on Luria-Bertani (LB) medium and incubated in the dark at 30°C [31, 35].

#### **3.1 Bacillus**

*Bacillus* and its products have been known for application as biological control agent against a range of plant pathogen. The success of *Bacillus* species as biocontrol agent could be ascribed to a wide array of peptide antibiotics produced such as iturin A, mycobacillin, subtilin and bacilysin as well as 25 different basic chemical structures with proven antifungal secondary metabolites [36, 37]. Lamsal et al. [38] found after a dual culture inhibition assay was conducted on V8-PDA in plastic petri plates (8.5 cm diameter) that seven bacterial isolates (AB05, AB11-AB15 and AB17) qualified previously as beneficial microbes of tomato plants, inhibit efficiently *P. infestans* affecting tomatoes in Korea by more than 60% in vitro. However, AB15 was the most effective, inhibiting mycelial growth of the pathogen by more than 80% in vitro. For greenhouse evaluation, targeted plants were left to dry for 2 days, and then 100 ml of bacterial spore solution (107 spores/ml) was added to each pot 7 days before infection so that only soil, but no above-ground parts, received any bacterial spores. The results showed that AB15 was the most effective suppressing disease by 74% compared with control plants under greenhouse conditions. According to 16S rDNA sequencing, a majority of the isolates are members of *Bacillus*, and a single isolate belongs to *Paenibacillus*. In India, for *Bacillus subtilis* strains were tested for their biocontrol activity against *P. infestans* in presence of the fungicide (Mancozeb) M45 (CURZATE®) as positive control. Before the sowing of potato seeds in blocks, all blocks were drenched with different bacterial cultures at the concentration 2x106 CFU/ml, with the exception of chemical fungicide and control blocks. The potato seed tubers were treated with 0.2–0.3% of M 45 (Mancozeb) fungicide before ten days of planting. Results revealed that, bacterial treatments signifiantly reduced disease incidence of late blight compared with the control. Bacterial treatments increased the plant vegetative parameters like plant height, sprouting, number of leaves, fresh weight and dry weight of plants. In addition, treatments also showed the clear difference between commercial and non-commercial tuber yield/hectare. In a view of this results they suggest that the mode of the action were the ability of bacillus subtilis strains to produce mycotoxins which can inhibit *P. infestans* growth and the capacity of bacillus to induce the peroxidase activity [39]. Elliott et al. [40] have been reported that Companion® and Serenade® are two *Bacillus subtilis* commercial biocontrol products which reported to suppress *P. infestans*. However, resistance to this bioproducts develops and some isolates of *P. ramorum* from North American and European population have been shown to be resistant [41]. Bacillus strains could control *P. infestans* directly by inhibiting the mycelial growth, germination of the cysts or the swimming of the motile zoospore by producing many antifungal compounds which suppress the pathogen or indirectly mechanisms by inducing the inhabitation of the activity of ribosome or stimulate active oxygen burst, NO production, callose deposition, and lignification [42–44].

#### **3.2** *Pseudomonas*

Among biocontrol agents of interest, Pseudomonas spp., are known for their production of antibiotics involved in biocontrol, such as

#### *Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*

2,4-diacetylphluoroglucinol and phenazines [45–47], which have been widely studied in various plant-pathogen systems. Phenazine-1-carboxylic acid (PCA) producing Pseudomonas spp., have been found effective against numerous phytopathogens, including bacteria, fungi, and oomycetes, such as the causal agent of bacterial blight of rice, *Xanthomonas oryzae* pv. oryzae [48], *Gaeumannomyces graminis* var. tritici [49] and the oomycetes *Phytophthora* spp., and Pythium spp., [50, 51]. PCA has been linked to biofilm formation, favoring attachment of PCA-producing Pseudomonas spp., to plant roots which facilitate the role of this beneficial microbes as biocontrol agents [52]. The mechanisms involved to control *P. infestans* by Pseudomonas were recently investigated, a previous study by [53] reported that the biocontrol of the pathogen could be by inhibiting sporangia and zoospore germination which suggesting the presence of many yet unknown antioomycete determinants. However, [54] suggests that Phenazine-1-carboxylic PCA produced by *Pseudomonas spp*., is involved in *P. infestans* growth repression and led to important transcriptomic changes by both up and down regulating gene expression in *P. infestans* over time. Different metabolic functions were altered and many effectors were found to be upregulated after the application of PCA, suggesting their implication in biocontrol. The cyclic lipopeptide surfactant massetolide A is a metabolite with versatile functions in the ecology of *Pseudomonas fluorescens* SS101 [55]. To study the effects of *P. fluorescens* SS101 and massetolide A on late blight of tomato, two leaves located on the second branch from the stem base of 5-week-old tomato plants were immersed in bacterial suspension (109 CFU ml−1) for 1 min or in a solution of massetolide A in sterile demineralized water (pH 8). Leaves immersed in sterile demineralized water (pH 8) for 1 min served as a control. Treated tomato plants were transferred to trays covered with transparent lids. After incubation for 1 d in a growth chamber at 15°C, the lower side of each treated tomato leaf was inoculated with 3 μl droplets of a *P. infestans* zoospore suspension (3–4 × 103 swimming zoospores ml−1) or 3 μl droplets of sterile demineralized water (pathogenfree control). *P. fluorescens* SS101 was effective in preventing infection of tomato (*Lycopersicon esculentum*) leaves by *P. infestans* and significantly reduced the expansion of existing late blight lesions. Massetolide A was an important component of the activity of *P. fluorescens* SS101, since the massA-mutant was significantly less effective in biocontrol, and purified massetolide A provided significant control of *P. infestans*, both locally and systemically via induced resistance [56]. Additionally, Biosurfactants (Rhamnolipids) produced by *fluorescent Pseudomonas* have zoospore lysis activity and biosurfactant-producing strain *Pseudomonas koreensis* 2.74 has potential to induce resistance in potato plant against late blight disease. High sensitivity of *P. infestans* zoospores to biosurfactants suggest that they can be used to dampen the spread of potato late blight once infection has been detected in the field [57, 58].

#### **3.3** *Actinomycetes*

Actinomycetes are Gram+ bacteria that represent a high proportion of the soil microbial biomass and have the ability to produce a wide variety of antibiotics and of extracellular enzymes. Several strains of actinomycetes have been found to control plant diseases [59–61]. Recently, [62] were identifed β-rubromycin as a *P. infestans* cyst germination inhibitor by screening compounds produced by *Streptomyces* isolated from soil. For that, an acetone extract was prepared from *Streptomyces* cultures grown for 5 days in liquid medium A at 30°C by adding an equal volume of acetone followed by mixing. 20-μL aliquots were mixed with 1 × 103 *P. infestans* sporangia in total 70 μL (14.2% acetone solution), incubated at 10°C for 18 h, and examined using an inverted microscope. As a control, it is

confirmed that 15% acetone had no effect on morphological change in *P. infestans*. The isolation of the cyst germination inhibitor enabled to identify β-Rubromycin which can inhibit *P. infestans* cyst germination and hyphal elongation from sporangia, while not affecting zoospore release, cyst formation, or appressorium formation. Chemical genetic analyses using β-rubromycin identifed a RIO kinase-like gene, PITG-04584, as a critical contributor to zoosporogenesis, cyst germination, and the formation of appressoria in *P. infestans*. The Lubimin is a vetispirane sesquiterpenoid that consists of (2R,5S,6S,8S,10R)-8-hydroxy-10-methyl-2-(prop-1-en-2-yl)spiro[4.5]decane bearing a formyl substituent at position 6. It has a role as an antifungal agent and a phytoalexin. The synthesis of this biocompounds in noninoculated potato tuber slices have been elicited after using culture filtrates of *Streptomyces* isolates which induce the resistance of potato plants against late blight caused by *P. infestans* [63]. In this sense, the reliance on actinomycetes as promising biocontrol strategies are very useful in controlling *P. infestans*. Several actnimoycetes most of which were *Streptomyces* strains have been demonstrated to be effective [64–67].

From the gastrointestinal tract of a fish dredged near the South Orkney Islands in Antarctica, [68] isolated the psychrotolerant bacterial *Vibrio splendidus* T262. Investigation of this strain led to the isolation of a rare series of 15 bis- and trisindole derivatives. Among them, six new indole alkaloids. Using the agar diffusion method, at 10 μg/paper disk, some of the isolated compounds showed activity against both gram-positive and gram-negative bacteria when trisindolal was active against the *P. infestans* and a number of other plant-pathogenic fungi.

Independently of the mode of action of biological control agents, the successful application of rhizobacteria to suppress late blight was confirmed by several research using a range of bacteria such as *Micrococcus luteus*, *Paenibacillus* spp., *Flexibacteraceae bacterium*, and *Enterobacter cloacae* [35, 40, 69, 70]. However, there is a lack of research that highlight the effectiveness of the combination assays of one or more bacteria to control *P. infestans*. Whereas, the combinations have potential for extensive colonization of the rhizosphere, more consistent expression of beneficial traits under a broad range of soil conditions, and antagonism to a larger number of pathogens than strains applied individually.

#### **4. Fungi**

The beneficial fungi have gained immense attention as biofertilizers due to their role in maintaining plant quality and quantity and their environmentfriendly relationship. Nowadays, use of this microorganisms as biocontrol agent (BCA) is considered to be a rapidly developing natural phenomenon in research area. Fungal biocontrol agents (BCAs) do not cause any harm to the environment, and they generally do not develop resistance in various types of pathogens due to their complex mode of action. They have been proved to be an alternative against the undesirable use of chemical products [71–73]. Previous reports have detailed the importance of various fungi species as effectiveness biocontrol agents against *P. infestans* [74–76]. For beneficial fungi isolation, the same method was adopted for years ago based on PDA medium and it can have same small modifications. PDA with chloramphenicol 0,016% (PDAc) and Rose Bengal Agar (RBA) (dextrose 10 g.l−1, meat peptone 10 g.l−1, K2HPO4 1 g.l−1, MgSO4.7H2O 0.5 g.l−1, Rose Bengal 30 mg/l, Agar 20 g/l) media were used. Petri dishes were incubated for 4 days for bacterial isolation and 7 days for fungal isolation at 25°C in the dark [59, 77].

*Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*

#### **4.1** *Trichoderma*

In the thick of various beneficial microbes have been investigated by several scientists, Trichoderma genera is a well-known biocontrol fungi that has been used since the 1930s to help plants acquire nutrients and control the plant pathogens [78]. Several Trichoderma species have been developed commercially as biofungicides and biofertilizers.

Fungi in the genus *Trichoderma* and bacteria such as *Bacillus amyloliquefaciens* have shown in vitro potentiality to reduce the mycelial growth of *Phytophthora infetsans*, *P. quercina*, *P. capsici*, *P. cactorum* and P. plurivora attacking *Quercus robur*, *Fagus sylvatica* and *Capsicum annuum* [35, 79, 80]. The biocontrol roles of *Trichoderma* against *P. infestans* could be attributed to the Trichoderma's rhizosphere competence and competitive ability [81], via the use of many mycoparasitic strategies which are a direct mechanism for biological control that works by parasitizing, detecting, growing, and colonizing pathogen involving the detection of pathogens through chemotropism; lysis of the pathogen's cell wall, pathogen's hyphal penetration by appresorial formation; production of cell wall-degrading enzymes (CWDEs) and peptaibols and parasitizing pathogen's cell wall contents [82], antibiosis or by activating a defense response as well as increased plant growth [83]. Many studies have shown the biocontrol activity of Trichoderma against *P. infestans*. Khan et al. [84] reported for the first time the elucidation and production of viridiofungin A (VFA) from *T. harzianum* isolate T23 cultures and the antifungal potential of VFA against *P. infestans* by suppressing zoosporangia germination and exhibiting a high activity on germ-tube growth. In the assay, 0.3 ml PDB/V8 medium in 0.6 ml Eppendorf tubes containing VFA concentrations from 50 to 200 μg ml−1 and sporangial suspensions of the pathogen were prepared. Control medium contained 2% acetone. Cultures were incubated on a shaker at 100 rpm at 25°C in the dark for 24 h. Subsequently, aliquots were taken from the cultures. Germination rates of sporangia and germ tube elongations were determined. Moreover, [85] highlighted the ability of 14 strains of *Trichoderma* to emit volatile compounds that decreased or stopped the growth of *P. infestans*. The experiments were performed in Petri plates divided into two compartments. The first compartment, containing V8 agar, was inoculated in the center with a 5 mm diameter mycelial disk of *P. infestans*. The second compartment, containing PDA, was inoculated with 5 mm mycelial disk of actively growing mycelia from one of the 14 *Trichoderma*. The plate-dividing wall prevented any physical contact between the *Trichoderma* strains and *P. infestans* but allowed the free exchange of VOCs. After inoculation, the plates were sealed with two layers of Parafilm and incubated at 21°C for 6 d, at which point the growth diameters were recorded. Volatile organic compounds (VOCs) emitted from *Trichoderma* strains inhibited the mycelial growth of *P. infestans* grown on a laboratory medium by 80% and on potato tubers by 93.1%. Using GC–MS analysis showed that the most abundant compounds were 3-methyl-1-butanol, 6-pentyl-2-pyrone, 2-methyl-1-propanol, and acetoin. Electron microscopy of the hyphae treated with *T. atroviride* VOCs revealed serious morphological and ultrastructural damages, including cell deformation, collapse, and degradation of cytoplasmic organelles.

#### **4.2** *Penicillium*

Large number of reports mentioned that *Penicillium* spp., interact positively with plants roots. Some *Penicillium* species have shown an antagonistic activity against plant pathogens by producing antibiotics which is a primary mechanism of disease suppression by *Penicillium* also induce resistance in plants by activating defense signals [86, 87]. The adaptability to different environments and tolerance to various abiotic stresses gives theses fungi species an advanced ranking to suppress many plant pathogens [87]. Previous reports have demonstrated that *Penicillium* species show efficacy as biocontrol agent against *P. infestans*. Based on the study conducted by [77] reported that *Penicillium chrysogenum* induce resistance against *P. infestans* in tomato plants. Dry *Peni. chrysogenum* mycelium extract was prepared using a detailed protocol described by [77] the extract was diluted with distilled water to a total carbohydrate content of 1.5 g l−1. The tomato plants were treated two times with about 25 mL extract per plant as foliar spray. Leaf discs (diam. 18 mm) of plants treated were laid onto moist filter paper. Leaf discs were inoculated with 10 L droplets of zoospore suspension. The inoculated leaf material was kept at 23°C in the dark with a relative humidity at 100%. Three days later biochemical assays for the peroxidase activity and isoenzyme analysis were conducted. The application of the water extract of killed *Peni. chrysogenum* has shown no direct antifungal activity against the pathogen, however the protective effect of the extract was shown under controlled conditions after application on the whole plants and on leaf disk. The findings suggest that control resulted from the induction of defense mechanisms in the tomato plants. According to this many reports have been shown that the ability of *Penicillium* species to induce systemic expressions of defense-related genes [peroxidases (POX) and phenylalanine ammonia lyase (PAL) and PR-1 genes] is the key used by *Penicillium* to induce plant defense systems as well protects plants from pathogens [85, 86]. Otherwise, antagonistic activity of endophytic fungi associated with *Artemisia nilagirica* was studied against the pathogen *P. infestans* by the presence or absence of inhibition zone observed in dual cultures by using dual culture methods. The study has shown that among the endophytic fungal tested *Penicillium atrovenetum* and *Trichoderma viride* showed direct inhibition activity of pathogen mycelia growth [87]. Additionally, [88] reported that *P. striatisporum* Pst10 isolated from the rhizosphere of chili peppers showed very high antagonistic effects on mycelium growth and sporangia/spore formation or germination of *Phytophthora* spp., The analysis of the Pst10 organic solvent extract by thin-layer chromatography (TLC) and the antagonistic activity tests highlight the existence of three different antifungal compounds produced by *P. striatisporum* Pst10. To study the Pst10 antifungal spectrum of Pst10 the dual culture assays were used. In the other hand, To determine the effect of Pst10 sterilized liquid culture filtrates (SLCF) on sporangium and spore germination, 100 μl of sporangium or spore suspensions of *P. capsici* were spread on 20 ml PDA agar containing 1 ml Pst10 SLCF. PDA plates were incubated at 28 C for 24, 72, and 120 h. After each incubation time, 100 sporangia or spores were counted and germination rate was calculated under a light microscope.

Using fungi as biological agents to control or suppress the growth of *P. infestans* is not just limited to *Trichoderma* and *Penicillium* even they were the most fungi reported. In 2020 [75] Isolated *Aspergillus flavipes* from agricultural soils as a strong inhibitor for growth of various species of *Phytophthora*. As well as, the crude extracellular extract of broth cultures of *A. flavipes* displayed a significant growth inhibition of various *Phytophthora spp*., The putative compounds from *A. flavipes* were chemically verified as 3-hydroxy-2′,4,4′,6′-tetramethoxychalocone, 7,3,4,5′-tetramethoxyflavanone, isovitexin and amodiaquine. The non-activity of this compounds on several pathogens while their noticeable drastic effect on *Phytophtora* zoospores germination, mycelial anastomosis, sporangial formation and causing enlarged hyphal tips, dwarfness to the hyphal length. This results suggest that *A. flavipes* compounds are considered potentially as antiphytophthoral. Moreover, [89] described an antifungal metabolite, oosporein, which was isolated from the liquid culture of *Verticillium psalliotae* that produced the antagonistic effects on *P. infestans*. Oosporein exhibited a significant growth-inhibitory effect

*Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*

on *P. infestans* in comparison with other phytopathogenic fungi. De Vries et al. [90] found that Out of an analysis of 12 fungal endophytes, *Phoma eupatorii* isolate 8082 and *Monosporascus* spp., inhibited the growth of *P. infestans* in co-culture using the agar diffusion assays, co-inoculation in planta and anthocyanin, presumably through the secretion of secondary metabolites, particularly since their culture extracts were also active. Furthermore, the study reported that the two of the endophytes exhibited global inhibition of nine European *P. infestans* isolates. These examples indicate that many fungi species as a beneficial microbes are also characterized with high potential to control *P. infestans* directly by antagonistic activity which inhibit the mycelia growth and the zoospore/zoosporangia germination via the production of a range of biocompounds and by the induction of defense mechanisms. Nonetheless, the use of beneficial fungi as a potential candidate to be more studied and tested as a novel biocontrol agent in the field providing an alternative to resistance gene breeding and application of agrochemicals.

#### **5. Mode of action**

As mentioned early, previous investigations highlight the importance of fungi and bacteria as biological control against *P. infestans*. Thus, gaining insight into mechanisms is of high importance for disease control. It is reported that microorganisms engage several antagonistic mechanisms against plant pathogens, including antibiosis, mycoparasitism, competition for nutrients and space, promotion of plant growth, induced plant defense mechanisms, and modification of environmental conditions. Among those mechanisms, the antibiosis refers to interaction lethal between microorganisms through secondary metabolites, which is of high importance to identify target cell, protein or enzyme, in concrete, implicated in the mechanism. Moreover, identification of chemical substance responsible on inhibiting of plant pathogens is a task challenge, due to volatility of compounds and their synergetic effects. Until now, fewer compounds from microorganisms were shown to effectively affect *P. infetans*. These include Phenazine-carboxylic acid [91], Oosporein [92], β-Rubromycin [93], Iturin A [94], Fenngycin A, [95, 96], Thiobutacin [97], Bikaverin [98], Fusaric acid [98], 2,5-diketopiperazine [99] and Xenocumacine 1 [100], listed in **Figures 1** and **2**. Moreover, detailed mechanism of interaction against *P. infestans* was developed only with β-Rubromycin, Iturin A and phenazine-1-carboxylic acid. β-Rubromycin belongs to the quinone antibiotics that have the ability to inhibit retroviral reverse transcriptase but also act as inhibitors of DNA polymerases [101]. [94] evaluated the activity of β-Rubromycin produced by *Streptomyces* isolated from soil against *P. infestans*, showing that this compound was capable of inhibiting the infection caused by sporangia and zoospores in tomato plants. The mechanism of action seems to be related to the up regulation of the RIO kinase-like gene that are involved in morphological development, altering processes as important in *P. infestans* as cyst germination and hyphal elongation. [95] evaluated the biocontrol capacity of *Bacillus subtilis* WL-2 against *P. infestans*, establishing that Iturin A was the metabolite involved in the inhibition capacity against this phytopathogen, causing cell membrane disruption and an irregular internal cell structure. Iturin A is a lipopeptide that exerts its antimicrobial action through the alteration of the cell membrane via the production of pores that generate osmotic perturbation [102]. In addition to its activity in the membrane it was observed that iturin A was capable of generating mitochondrial damage in *P. infestans*, causing oxidative stress and alterations in the respiratory chain which alter ATP synthesis. [54] reported the effect of phenazine-1-carboxylic acid (PCA) produced by a strain of *Pseudomonas fluorescens* on the transcriptome of *P. infestans*, establishing that this

#### *Agro-Economic Risks of* Phytophthora *and an Effective Biocontrol Approach*

**Figure 1.**

*Anti-*Phytophthora infestans *compounds produced by fungi microorganisms.*

#### **Figure 2.**

*Anti-*Phytophthora infestans *compounds produced by bacteria microorganisms.*

compound alters the expression of genes involved in functions like phosphorylation mechanisms, transmembrane transport and oxydo-reduction activities.

Another method of disease control, so-called Mycroparasitism, is able to antagonize plant pathogens and promote plant growth by treatment with other microorganisms. Mycoparasitism is a direct mechanism in which microogranism colonizes the

#### *Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*

pathogen through detection, parasitization and growth actions [103, 104]. This protection strategy has been recognized as an important mechanism of biological control. Mycoparasitics such as the oomycete *Pythium oligandrum* [105], *Pythium periplocum* [106] and different species from *Trichoderma* including *T. asperellum*, *T. atroviride*, *T. virens*, and *T. harzianum* are successfully used against *P. infetans*. These mycoparasitic grow faster than their pathogenic plant counterparts, which means that they can occupy rhizosphere space and nutrition, thus promoting both plant growth [107] resistance in host plants [108, 109]. The mechanism of Trichoderma spp., for example, appear to be very complex involving the detection of plant pathogen through chemotropism; lysis of the pathogen's cell wall (the key to mycoparasitism) [110]; pathogen's hyphal penetration by appresorial formation; production of cell wall-degrading enzymes (CWDEs) and peptaibols, mediated by heterotrimeric G-proteins and mitogen-activated protein (MAP) kinases [111]; and parasitizing pathogen's cell wall contents [112]. Degradation of pathogen's cell wall during mycoparasitism is mediated by a set of hydrolytic enzymes including β-(1,6)-glucanases, chitinases, and proteases. Several members from each of these classes have been shown to be involved in mycoparasitism and/or to be induced under mycoparasitism-related growth conditions [113]. Although these microorganisms demonstrate their potential as mycoparasitic biological control agents, fewer mechanistic studies have been investigate the molecular or genetic determinants of their mycoparasite lifestyle.

However, rather than directly expanding into infected plant, microorganism might compete with the pathogen producing secondary metabolites able to partially or totally inhibit the pathogenic fungi. This classical mechanism occurs when special and nutritious resources are limited. Consequently, the antagonistic microorganisms feed on the available resources for growth, causing therefore a reduction in the growth of the pathogens. A published example of metabolitepathogen protection is that produced by *Phoma eupatorii* 8082. This endophyte has a remarkable potential to produce the anthocyanin product [114]. The latter could be produced as a result of a stress response positively regulated by jasmonic acid [115–118]. Hence, it is possible that tissue colonization with *Pho. Eupatorii* induce jasmonic acid dependent defense responses, which may play a role in the inhibition of the *P. infestans* infection. Indeed, [119] reported that jasmonic acid induced reduction of infection in the leaves of tomato and potato plants and [120] testified the mandatory existence of jasmonic acid to activate the defensive responses elicited by a peptide secreted by *P. infestans*. Some microorganisms including *Trichoderma* spp., produce inorganic compounds able to alter soil pH and therefore able to modify micronutrients (phosphate, iron and Manganese) [121]. In these extreme conditions *Trichoderma* spp., were able to produce various kinds of Siderophore products [122], including: caprogens, ferrichromes and fusarinines [123], thanks to the change in non-ribosomal peptide synthetase products and diverse nonribosomal peptide synthetase-encoding genes [124]. Siderophores play a dual role, an antagonistic agent by inhibiting or even suppressing the growth of pathogens by divesting source of iron, as well as an agonist agent that helps to solubilize iron that was not available to the plant. These abilities explain the competition mechanism on the nutrient resources.

Alternative mechanism of disease control against attack of pathogens is based on the induction of systemic and local resistances [125]. Such resistances result from complex interactions between plants and antagonist elicitors, provoking physiological and biochemical alteration of cells. Indeed, two major kinds of systemic resistances have been studied; systemic acquired resistance (SAR) [126] and induced systemic resistance (ISR) [127]. Both systemic resistances are based on distinct phytohormonal signals. Various compounds have been proposed as potential signals for systemic resistances activation. The non-protein amino acid, β-Aminobutyric acid

(BABA), is known to induce resistance against various pathogens on a wide range of plants. Indeed, DL- β-Aminobutyric acid-induced resistance of potato against late blight pathogen *P. infestans* trough the signaling compound salicylic acid [128]. BABA also provided significant control against *P. infestans* on tomato [129]. The systemic defense is induced in a salicylic acid dependent manner; furthermore various inorganic chemicals including indole acetic acid, di-potassium hydrogen orthophosphate, hydrogen peroxide, calcium chloride, ferric chloride and metalaxyl were able to induce resistance against the disease caused by *P. infestans*. Treatment with those agents promotes the synthesis of defense enzymes like peroxidase, polyphenol oxidase (POX) and phenylalanine ammonia lyase (PAL) [130]. In addition, Curdlan b-1,3-Glucooligosaccharides has shown to enhance plant resistance against the pathogen *P. infestans* in foliar tissues of potato (*Solanum tuberosum* L. cv. McCain G1) by accumulation of H2O2 and salicylic acid and the activities of phenylalanine amino-lyase, b-1,3-glucanase and chitinase [131].

### **6. Conclusion**

The application of beneficial bacteria and fungi as biocontrol agents is an interesting building block of sustainable and environmentally sound management strategies of *Phytophtora infestans*. A holistic approach should be considered to reach satisfactory levels of *P. infestans* control by a beneficial microbes. Based on the number of currently known isolates with biocontrol activity against *P. infestans*, the predominant genera are *Pseudomonas*, *Bacillus*, *Streptomyces*, *Trichoderma* and *Penicillium*. The ability to affect survival structures, sharing the same ecological niche as *Phytophtora*, inducing resistance responses in the plant and promoting plant growth are desirable characteristics of a competent BCA against *P. infestans*. However, among several criteria the potential bottlenecks such as large-scale production, formulation, preservation conditions, shelf life, application methods, and combination potentiality of one or more microbes should be tackled early in the selection process.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Appendices and nomenclature**


*Plant Beneficial Microbes Controlling Late Blight Pathogen,* Phytophthora infestans *DOI: http://dx.doi.org/10.5772/intechopen.99383*


#### **Author details**

Brahim Oubaha1 \*, Abdellah Ezzanad2 and Hernando José Bolívar-Anillo3

1 Laboratory of Microbial Biotechnologies Agrosciences and Environment (BioMAgE), Department of Biology, Faculty of Sciences Semlalia, University of Cadi Ayyad, Marrakech, Morocco

2 Department of Organic Chemistry, Faculty of Sciences, University of Cadiz, Cadiz, Spain

3 Laboratorio de Investigación en Microbiología, Facultad de Ciencias Básicas y Biomédicas, Universidad Simón Bolívar, Barranquilla, Colombia

\*Address all correspondence to: oubaha123@gmail.com; brahim.oubaha@edu.uca.ac.ma

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**Chapter 4**

## Endophytic Microorganisms as an Alternative for the Biocontrol of *Phytophthora* spp.

*Hernando José Bolivar-Anillo, Victoria E. González-Rodríguez, Giovanna Reyes Almeida, Inmaculada Izquierdo-Bueno, Javier Moraga, María Carbú, Jesús M. Cantoral and Carlos Garrido*

#### **Abstract**

The genus *Phytophthora* with more than 100 described species and 58 officially recognized, phylogenetically distributed in ten clades, are important pathogenic oomycete chromists that cause important diseases in agricultural crops, trees and forests worldwide. This genus is known as "The Plant Destroyer" which causes great economic losses with costs between 2 and 7 billion dollars per year in agricultural systems and unquantifiable losses in natural ecosystems. The host plants of the genus *Phytophthora* can vary from a wide range in some species to only one host, however, the host plants of the new species are still being determined and therefore the range continues to expand, that makes control exceedingly difficult. Plant damage can range from alterations in roots, fruits, trunks, stems, foliage and crown to invasive processes in highly susceptible species. Considering the wide range of hosts and organs that can be affected by *Phytophthora*, the use of endophytic microorganisms for the biocontrol of this phytopathogen can be an alternative to avoid losses of both crops and forests worldwide. Endophytes are microorganisms that live inside plant tissues without causing disease under any circumstances. The fact that endophytic microorganisms are able to colonize an ecological niche similar to that of some plant pathogens qualifies them as potential biocontrol agents. This chapter describes the endophytic bacteria and fungi isolated from different plant species that have shown antagonistic activity against different species of *Phytophthora*, as well as the metabolites isolated from these microorganisms that have shown fungicide activity and other biocontrol strategies (enzyme production, siderophores, substrate competition, among others) against *Phytophthora*.

**Keywords:** biological control agents, biocontrol, inhibitory mechanims, endophytic fungus, bacteria

#### **1. Introduction**

Phytopathogenic microorganisms are one of the main factors to causes losses in yield and quality of the crop along the world (worldwide). The economic losses due to diseases caused by microorganisms during pre- and post-harvest has been estimated to be between 30–40%, reaching almost 40 billion dollars worldwide annually [1, 2].

There is a great biodiversity of microorganisms that can cause diseases in plants. The group formed by phytopathogenic oomycetes (fungal-like organisms), is one of the most important and oldest. They have affected humankind since the beginning of agriculture in early civilisations [3]. During the last few centuries, these pathogens were responsible for the Potato Famine in Ireland, also known as the Great Famine, which caused almost a million deaths and triggered a mass migration in 1840 in that country [4, 5]. Even today, *Phytophthora infestans* is the causal agent of this disease in potatoes, it being the most important biotic limitation for the production of this tuber worldwide [4]. Other species of oomycetes, such as *Phytophthora ramorum*, do not only affect agriculture but also the environment, as they cause several diseases in many species of trees. As a consequence of the loss of forest mass due to infections and dead plants, it has been estimated an indirect impact on the environment that could reach a cumulative loss of 230–580 megatons of dissolved CO2 during the last century [3]. Currently, these phytopathogens continue to represent a significant danger in agricultural and forestry systems because they have accelerated their evolution. This is caused by the continued use of fungicides, together with dispersal dependent on anthropogenic activities and climate (i.e. natural aerial dispersal and climate change). The use of monocultures as well as the greater use of perennial crops also increase the sexual recombination events of the populations of these oomycetes [4, 6]. This could cause an adaptation and improvement of these pathogens that would allow them to expand the range of hosts [4].

Among the phytopathogenic oomycetes, those of the genus *Phytophthora* are the best studied [1]. The genus *Phytophthora* is presently placed in the kingdom *Straminipila*, phylum *Heterokonta*, sub-phylum *Pernosporomycotina*, class *Pernosporomycetes* (Oomycetes), subclass *Pernosporomycetidae*, order *Pythiales* and family *Pythiaceae* [7]. *Phytophthora* has more than 100 described species and 58 officially recognized, phylogenetically distributed in ten clades, are usually soil-borne plant pathogens that cause important diseases in agricultural crops, trees and forests worldwide [8, 9]. This pathogen can present biotrophic, necrotrophic, or hemibiotrophic lifestyles [1, 3]. They reproduce asexually giving rise to sporangia, which divide into zoospores. When conditions are favourable, zoospores germinate to form mycelia or a specialized infection structure called appressorium. Sporangia can also germinate directly to produce mycelia or form an appressorium. Both sporangia and zoospores are important cells in the dissemination and infection processes [1].

Among the crops that can be infected by the genus *Phytophthora* are potato, tobacco, soybean, avocado, macadamia, cocoa, rice, tomato, pistachio, red pepper, strawberry, raspberry, among others [9–12]. Natural vegetation and ornamentals can also be infected by *Phytophthora* species, i.e. oaks, alder, holm, chestnut, cork oak, beech, rhododendron, viburnum, magnolia, pieris, among others [11–14]. Some species are highly specific to the host (i.e. *P. sojae*) or with a wide range of possible hosts (i.e. *P. cinnamomi*). However, the host plants of the new species are still being determined and, therefore, the range continues to expand, making control exceedingly difficult [7, 11]. Plant damage can range from alterations in roots, fruits, trunks, stems, foliage and crown to invasive processes in highly susceptible species [8, 9].

The control of infections caused by *Phytophthora*, in agriculture, forestry and natural systems is very limited. The fungicides available are usually not efficient against oomycetes since they are not true fungi [11]. Furthermore, the use of chemical fungicides is being increasingly restricted due to the adverse effects they produce on human, animal and environmental health [15]. As an alternative to the use of chemical products, the idea of using antagonistic microorganisms or the metabolites that they produce is proposed for the biocontrol of these oomycetes. The microorganisms used for biocontrol do not have negative effects on human or animal health and are considered friendly with the environment. Biocontrol carried

*Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

out by microorganisms offers multiple modes of action, both direct, indirect or mixed, in addition, it prevents the appearance of resistance, which makes them an attractive alternative or complement for the control of phytopathogens [16]. The ability to biocontrol diseases through the use of microorganisms highlights the importance of interactions between the plant, the pathogen, the antagonist, the microbial community associated with the plant and environmental conditions [17, 18]. In this sense, most of the microorganisms used in biological control have been isolated from areas related to plants such as the rhizosphere, endosphere, phyllosphere, spermosphere, among others [19]. Although rhizosphere microorganisms are the most used in biocontrol, in recent decades a considerable number of endophytic microorganisms have been studied for their ability to biocontrol and for being a new source of natural products for use in agriculture [18, 20–22]. Therefore, this chapter describes the endophytic bacteria and fungi isolated from different plant species that have shown antagonistic activity against different species of *Phytophthora*, as well as the metabolites isolated from these microorganisms that have shown fungicide activity and other biocontrol strategies (enzyme production, siderophores, substrate competition, among others) against *Phytophthora*.

#### **2. Endophytic microorganisms as a biocontrol strategy**

Endophytes are microorganisms that are found inside plant tissues during at least part of their life cycle. They do not cause disease under any circumstances, and many

**Figure 1.** *Mechanisms of biocontrol showed by endophytic microorganisms.*

show properties that promote plant growth [23, 24]. Approximately 300,000 species of plants have been described, and it is believed that each may possess different genera and species of endophytic microorganisms. However, it has only been studied the endophytic microbiome of 1–2% of plants. There are many unexplored fields of research on endophytes and their potential as biocontrol agents [25–28]. Although most endophytes are considered commensals, a large number of them establish mutualistic relationships with their host plant, playing a fundamental role in the adaptation of plants to biotic and abiotic factors [29–32]. Their use as biocontrol agents is considered one of the main characteristics to be used in the control of phytopathogens in agriculture. In this way we could reduce or avoid the use of antimicrobial compounds of chemical origin [18]. Endophytes can exert their biocontrol activity through various mechanisms including competition for a niche or substrate, hyperparasitism, predation, allelochemical production (antibiotics, lytic enzymes, siderophores) and by inducing systemic resistance in plants (**Figure 1**) [26, 33]. Now, the efficiency of endophytes as biological control agents depends on factors such as the specificity of the host, the physical structure of the soil, environmental conditions, the growth phase and the physiological state of the plant, among others [18, 34]. The development of a disease in a plant by any phytopathogenic microorganism will depend on three factors: the plant-the microbiota-the pathogen, whose interaction will be influenced by environmental factors. The loss of balance in any of these three factors would therefore lead to the development of an infectious process or not. On the other hand, most endophytic microorganisms originate in the soil (rhizosphere), therefore their recruitment (by the plant) will depend on their existence in soil, which is because they are not always present [35].

#### **3. Biocontrol of** *Phytophthora* **spp. by endophytic bacteria**

The promotion of plant growth by endophytic bacteria can be carried out through direct or indirect mechanisms [26, 36]. Among the indirect mechanisms, there is the biological control of phytopathogens, which is carried out through various strategies such as competition for nutrients and space, antibiosis, production of lytic enzymes, inhibition of toxins and induction of defense mechanisms in plants. All these strategies can be compatible with each other and may co-act simultaneously or synergistically [16, 18, 26, 37]. In this regard, there have been various studies that have evaluated the potential of endophytic bacteria for the biocontrol of different species of *Phytophthora*. These bacteria have been isolated from different plant species, which has led to the identification of microorganisms and the mechanisms used by them to inhibit the growth of this oomycete. **Table 1** shows some endophytic bacteria isolated from different plant species and the possible mechanisms they use for the biocontrol of *Phytophthora* spp.

El-Sayed *et al.,* (2018) [38] isolated forty morphologically distinct bacterial from roots, stems and leaves of *Smilax bona-nox* L. and they belonged to the genera *Burkholderia, Pseudomonas, Xenophilus, Stenotrophomonas, Pantoea, Enterobactriaceae, Kosakonia, Microbacterium, Curtobacterium, Caulobacter, Lysinibacillus* and *Bacillus*. Out of these isolates, the ones that showed the highest *in vitro* growth inhibition capacity of 5 species of *Phytophthora* (*P. parasitica, P. cinnamomi, P. palmivora, P. tropicalis* and *P. capsici*) were two strains of *Pseudomonas fluorescences* (EA6 and EA14). The percentage of inhibition of mycelial growth against different strains of *P. parasitica* was between 47% and 80%. On the other hand, the crude proteins (extracellular hydrolytic enzymes) obtained from *P. fluorescence* EA6 were able to inhibit the mycelial growth of *P. parasitica*. The analysis of these proteins revealed that they were glucanolytic enzymes (β-1,3 and β-1,4 glucanases) which act by

**Microorganisms Plant species Inhibitory mechanisms Ref.** *Pseudomonas fluorescences Smilax bona-nox* L Glucanolytic enzymes [38] *Burkholderia* spp. *Huperzia serrata* Siderophores [39] *Acinetobacter calcoaceticus Glycine max L.* Siderophores [40] *Bacillus cereus Lycopersicon esculentum* Triggering the plant immune defense [41] *Bacillus Paenibacillus Lactococcus Pediococcus Enterobacteriaceae Cronobacter Pantoea* Seeds Cucurbits Antibiosis VOCs RNase activity [42] *Streptomyces Microbispora Lens esculentus Cicer arietinum* L. *Pisum sativum Vicia faba Triticum vulgare* Antibiosis Siderophores [43] *Bacillus thuringiensis B. vallismortis B. amyloliquefaciens Cornus florida Carica papaya* Antibiosis Triggering the plant immune defense [44] *Pseudomonas putida Piper nigrum* VOCs [45] *Streptomyces deccanensis Bacillus* spp. *Rhizobium radiobacter Pantoea dispersa Bacillus velezensis Acinetobacter* spp. *Piper colubrinum* Competition Antibiosis Triggering the plant immune defense [46] *Streptomyces alboniger Pseudomonas Dodonaea viscosa* Antibiosis [47]

#### *Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

*taiwanensis P. geniculata Enterobacter hormaechei Bacillus tequilensis B. flexus Arthrobacter phenanthrenivorans Delftia lacustris Fagonia indica Caralluma tuberculata Calendula arvensis* VOCs Cell wall degrading enzymes Siderophores *Bacillus megaterium Piper nigrum* VOCs HCN Hydrolytic activity Siderophore [48] *Pseudomonas aeruginosa Chryseobacterium proteolyticum Theobroma cacao* VOCs Hydrolytic activity Siderophore HCN [49] *Bacillus velezensis Olea europaea* Antibiosis VOCs Cell wall degrading enzymes Siderophores [50] *Alcaligenes* spp. *Hevea brasiliensis* PCA [51] *Bacillus siamensis B. amyloliquefaciens B. velezenis B. methylotrophiycus Piper nigrum* Cell wall degrading enzymes Antibiosis [52]

#### **Table 1.**

*Endophytic bacteria able to biologically control* Phytophthora spp*.*

hydrolyzing the cell wall of *Phytophthora*. In addition, the crude glucanolytic extract was shown to have higher activity than the purified β-1,3-glucanase enzyme, which means that these enzymes act synergistically on the cell wall of *Phytophthora*. Want *et al.,* (2010) [39] from *Huperzia serrata*, isolated the endophytic bacteria identified as *Burkholderia* spp. H-6, which was able to inhibit the *in vitro* mycelial growth of *Phytophthora capsici* with a diameter of inhibition zones of 23 mm. Furthermore, in greenhouse pot experiments, the soils treated with *Burkholderia* spp*.* densities of 106 , 108 and 1010 CFU ml−1 reduced *P. capsici* infection in pepper seedlings by 51.7, 58.7 and 60.2%, respectively. This strain presented the ability to synthesize siderophores, which could be related to its biocontrol capacity. Zhao *et al.,* (2018) [40] isolated a total of 276 endophytic bacteria from *Glycine max* L. nodules, of which 6 had an inhibition capacity greater than 63% against *Phytophthora sojae* and were identified as *Bacillus cereus*, *Acinetobacter calcoaceticus, Enterobacter cloacae, Bacillus amyloliquefaciens, Pseudomonas putida* and *Ochrobactrum haematophilum.* The strain identified as *Acinetobacter calcoaceticus* DD16 was the one that presented the highest inhibition of mycelial growth of *P. sojae* with 71.14%. *A. calcoaceticus* DD16 caused morphological abnormal changes of fungal mycelia (e.g. lysis, formation of a protoplast ball at the end of hyphae, and split ends) that could be related to the production of anti-fungal substances and fungal cell-lysing enzymes. In addition, *A. calcoaceticus* DD16 was the strain that presented the highest capacity to produce siderophores (54.33 ± 0.093 μg mL−1) and was capable of fixing nitrogen and producing indole acetic acid, activities related to the promotion of plant growth. The regression analysis showed a significant positive correlation between siderophore production and inhibition ratio against *P. sojae*. Melnick *et al.,* (2008) [41] isolated from *Lycopersicon esculentum* a strain of endophytic bacteria identified as *Bacillus cereus* BT8, which *in vitro* test did not show the ability to inhibit the mycelial growth of *Phytophthora capsici*. However, this strain exhibited the ability to colonize *Theobroma cacao* seedlings and reduce the severity of *Phytophthora capsici* infection. The suppression of *P. capsici* was only observed in leaves which were not inoculated with the endophytic bacteria after colonization of the plant in other leaves, which suggests that the mechanism of suppression of the disease is through the induction of defense mechanisms in the plants (Induced Systemic Resistance) rather than antagonistic mechanisms. Khalaf *et al.,* (2018) [42] isolated a total of 169 bacterial endophytes from seeds of diverse cultivated cucurbits (*Luffa acutangula*, *Curcubita moschata*, *Curcubita pepo*, *Lagenaria siceraria*, *Citrullus lanatus*, *Cucumis melo* and *Cucumis sativos*), of which 26% (44/169) of isolates showed anti-pathogenic traits *in vitro* against *Phytophthora capsici*, of these 44 isolates, 16 were obtained from *Cucumis melo* seeds. These bacteria with activity against *P. capsici* belonged to the genera *Bacillus*, *Paenibacillus*, *Lactococcus*, *Pediococcus*, *Enterobacteriaceae*, *Cronobacter* and *Pantoea*. Of these microorganisms, those of the genus *Bacillus*, *Paenibacillus*, *Enterobacteriaceae* and *Pantoea* showed acetoin/diacetyl production (volatile organic compounds VOCs) and RNase activity *in vitro*, known to be implicated in triggering the plant immune defense. Therefore, these bacteria may control the phytopathogen directly (antibiosis) and/or indirectly (induction of host defense).

Misk and Franco (2011) [43] isolated thirty-six actinobacterial strains from different plants (root, stem and leaf), lentil (*Lens esculentus*), chickpea (*Cicer arietinum* L.), pea (*Pisum sativum*), faba bean (*Vicia faba*) and wheat (*Triticum vulgare*). Eleven of the isolates had antimicrobial activity against *Phytophthora medicaginis*, where ten of those isolates belonged to *Streptomyces* and one to *Microbispora*. The strains identified as *Streptomyces* spp. WRA1 and BSA25 were the most efficient as they significantly inhibited 100% and 85% *in vitro* of *P. medicaginis*, respectively, which showed a good capacity to produce siderophores. Furthermore, *in vivo* tests both strains (WRA1 and BSA25) significantly inhibited

*Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

*P. medicaginis* root rot compared to infected control. This inhibition capacity against *P. medicaginis* could be related to their antibiotic and siderophores production. Bhusal and Mmbaga (2020) [44] evaluated the biocontrol capacity of three endophytic bacterias *Bacillus thuringiensis* isolated from flowering dogwood stem; *B. vallismortis*; and *B. amyloliquefaciens* isolated from papaya stem against *Phytophthora capsici. B. amyloliquefaciens* was the most effective in suppressing *P. capsici* mycelial growth *in vitro* up to 46.62%, followed by *B. vallismortis* 45.95% and *B. thuringiensis* 27.59%. Under the greenhouse environment, *B. amyloliquefaciens* and *B. vallismortis* were most effective in suppressing *P. capsici* symptoms. Agisha *et al.*, (2019) [45] evaluated the antimicrobial capacity on phytopathogens of VOCs produced by the black pepper endophytic bacterium, *Pseudomonas putida*. Of the VOCs produced by *P. putida*, those identified as 2,5-dimethyl pyrazine; 2-methyl pyrazine; dimethyl trisulphide; 2-ethyl 5-methyl pyrazine; and 2-ethyl 3, 6-dimethyl pyrazine showed inhibitory activity (sealed plate method) against *Phytophthora capsici*. Among these VOCs, 2-ethyl-3, 6-dimethyl pyrazine was the most effective with an EC50, EC90 and EC95 of 66.1 μg cm−3, 244.8 μg cm−3 and 382.1 μg cm−3, respectively. In trials to evaluate the effect of VOCs against *Phytophthora* rot on black pepper shoot cuttings, 2, 5 dimethyl pyrazine, 2-ethyl 5-methyl pyrazine and 2-ethyl 3, 6-dimethyl pyrazine displayed reduction of lesion at 21 μg cm−3 and, 2-methyl pyrazine at 42 μg cm−3 with no signs of toxicity. While in the tests for fumigant activity of volatiles, dimethyl trisulphide demonstrated complete inhibition against *P. capsici* at a concentration of 6.25 μg cm−3, which demonstrated that these VOCs can be an alternative for the control of *P. capsici* infections. Kollakkodan *et al.,* (2020) [46] isolated endophytic bacteria from the roots, stem and leaves of *Piper colubrinum*. Seven of these isolates showed *in vitro* inhibition capacity against *Phytophthora capsici* with zones of inhibition between 2.4 and 5.8 mm, which were identified as *Streptomyces deccanensis*, *Bacillus* spp., *Rhizobium radiobacter*, *Pantoea dispersa*, *Bacillus velezensis* (PCSE8), *Bacillus velezensis* (PCSE10) and *Acinetobacter* spp. The maximum inhibition zone was produced by the two strains of *B. velezensis*. In leaf assay (leaves of black pepper), the highest suppression of the disease was presented by the strains identified as *Pantoea dispersa* and *Bacillus velezensis* (PCSE10), with percentages of 74% and 79%, respectively. The mechanisms of these endophytic bacteria which are responsible for the inhibition of *P. capsica* seem to be mainly related to competition, antibiosis and triggering of the plant's immune defence. Iqrar *et al.,* (2021) [47] isolated endophytic bacteria from medicinal plants, *Dodonaea viscosa*, *Fagonia indica*, *Caralluma tuberculata* and *Calendula arvensis*. Bacteria that exhibited biocontrol activity on screening assays (production of cell wall degrading enzymes and siderophores) were identified as *Streptomyces alboniger*, *Pseudomonas taiwanensis*, *Pseudomonas geniculata*, *Enterobacter hormaechei*, *Bacillus pheustrivo*, *Bacillus flexus* and *Delftiartiabacteris*. In the *in vitro* growth inhibition test against *Phytophthora parasitica*, the highest inhibition was presented by the bacterium identified as *P. taiwanensis* with 55%, as well as in the bipartite split-plate growth inhibition assays (VOCs) with an inhibition of 80%. In addition, the crude extracts from the culture of this bacterium presented an inhibition of 92% at a concentration of 400 μg mL−1 and the ethyl acetate extract presented an inhibition of 60%. The hyphae of *P. parasitica* subjected to these extracts showed alterations in their structure (convoluted, swollen nodes and abnormal growth of hyphae). The inhibition capacity of these endophytic bacteria on *P. parasitica* seems to be related to multiple mechanisms of action such as antibiosis, VOCs, cell wall degrading enzymes and siderophores. Munjal *et al.,* (2016) [48] isolated an endophytic bacterium identified as *Bacillus megaterium* from the black pepper root that was capable of inhibiting different phytopathogens *in vitro*, including *Phytophthora capsici*. This bacterium exhibited the ability to produce hydrogen cyanide (HCN),

protease, cellulase and siderophore. In VOCs' activity tests, it was observed a growth inhibition of *P. capsica* of 28%. These VOCs were mainly composed of 2,5-dimethyl pyrazine, 2-ethyl-3-methyl pyrazine, 2-ethyl pyrazine and 2-methyl pyrazine and they were able to inhibit individual mycelial growth by more than 60% at a concentration of 336 μg mL−1. Among these VOCs, the most effective was 2-ethyl-3-methyl pyrazine, which 100% inhibited the mycelial growth of *P. capsici* at a concentration of 168 μg mL−1. Therefore, the antagonistic activity of this bacterium is related to the ability to produce VOCs, HCN, protease, cellulase and siderophore. Alsultan *et al.,* (2019) [49] isolated 103 endophytic bacteria from cacao plants (leaves, branches and fruits) of which two that showed an 80% *in vitro* inhibition of *P. palmivora* and were identified as *Pseudomonas aeruginosa* and *Chryseobacterium proteolyticum*. While in the culture filtrate test, the inhibition percentages were 100% and 62% to *P. aeruginosa* and *Ch. proteolyticum*, respectively. In the volatile metabolites test, *P. aeruginosa* and *C. proteolyticum* strains showed an inhibition of pathogen growth of 61.88% and 60.94%, respectively. The VOCs produced by *P. aeruginosa* were identified as eicosane, hexatriacontane, tetratetracontane, trans-2-decenoic acid and 1-phenanthrenecarboxylic acid, 1,2,3,4,4α,9,10,10α-octahydro-1,4α-dimethyl-7-(1-methylethyl), while those produced by *C. zproteolyticum* were identified as eicosane, tetratetracontane, heneicosane, hexatriacontane and phenol 2,4-bis(1,1-dimethylethyl). Regarding the hydrolytic activity, these two strains were capable of producing cellulase, protease, pectinase and lipase. Only *P. aeruginosa* was able to produce siderophores and HCN. The inhibition capacity of both strains is related to the capacity to produce hydrolytic enzymes, VOCs, HCN and siderophores that can act individually or synergistically. Cheffi *et al.,* (2019) [50] isolated the endophytic bacterium identified as *Bacillus velezensis* from olive trees, which exhibited an inhibition ranged from 40 to 75% with oomycetes, including *Phytophthora ramorum*, *P. cactorum*, *P. cryptogea*, *P. plurivora* and *P. rosacearum*. Regarding its biocontrol capacity, *B. velezensis* presented the capacity to produce VOCs, among which ethylbenzene, phenylethyl alcohol, E-caryophyllene and cyclo (Leu-Pro) were detected. Through genome analysis, diverse secondary metabolite clusters were uncovered such as bacillomycin, amylocyclin, mersacidin, bacilysin, macrolactin, bacillibactin, bacillaene, surfactin, fengycin, dicidin, subtilin and locillomycin. The analysis of the culture extracts by means of LC–MS, detected the production of surfactin B, surfactin C15, plipastatin B1, Fengycin B, IX and XII. Furthermore, this strain was able to produce cell wall degrading enzymes (protease, chitinase and glucanase) and siderophores. All these metabolites could be responsible for the inhibition capacity of *B. velezensis* on these oomycetes. Abraham *et al.,* (2015) [51] isolated the endophytic bacterium identified as *Alcaligenes* spp. from *Hevea brasiliensis,* that presents antagonistic activity against *Phytophthora meadii*. By means of the spectrometric study of the culture supernatant of *Alcaligenes* spp., it was established that the compound identified as phenazine-1-carboxylic acid showed inhibition of *P. meadii* growth. The minimum inhibitory concentration of this compound against *P. meadii* was optimized at 5 μg mL−1. In addition, this compound presented zoospore-lytic activity, the structure of which was completely altered and lysis of the same occurred. The zoospores were not able to germinate when they were cultured in the presence of this compound. Ngo *et al.,* (2020) [52] isolated endophytic black pepper bacteria, of which six showed the ability to inhibit the growth of *Phytophthora* spp. by more than 60%. These bacteria were identified as *Bacillus siamensis*, *B. amyloliquefaciens*, *B. velezenis* and *B. methylotrophiycus*. These strains presented high chiti-nase and protease activities. In the *in vivo* test, the strains identified as *B. siamensis*, *B. velezensis* and *B. methylotrophycus* (EB.KN13) had the lowest rate of root disease (8.45–11.21%) and lower fatal rate (11.11–15.55%).

*Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

#### **4. Biocontrol of** *Phytophthora* **spp. by endophytic fungi**

Like bacteria, endophytic fungi can protect their host plant against both biotic and abiotic stressors; which are considered a rich source of bioactive metabolites [32, 53, 54]. Among the main mechanisms by which endophytic fungi prevent infections by phytopathogens are induced resistance, antibiosis, mycoparasitism, competition and extracellular enzymes [31, 32, 54]. **Table 2** summarizes the species of endophytic fungi with biocontrol capacity against *Phytophthora* spp. and the plant species from which they were isolated, revealing the wide diversity of endophytic fungi that can be used for the biocontrol of this phytopathogen. Hanada *et al.,* (2010) [55] evaluated the antagonistic capacity of endophytic fungi isolated from *Theobroma cacao* and *Theobroma grandiflorum* against *Phytophthora palmivora*.


#### **Table 2.**

*Endophytic fungi with biocontrol capacity against* Phytophthora spp*.*

A total of 103 endophytic fungi were isolated of which ~70% showed some degree of reduction in the disease severity in three cacao pods. Eight isolates from genera *Trichoderma*, *Pestalotiopsis*, *Curvularia*, *Tolypocladium* and *Fusarium* showed the highest level of activity against the pathogen. The possible responsible mechanisms for the ability to inhibit *P. palmivora* were related to the production of bioactive compounds. Mitchell *et al.*, (2010) [56] evaluated the ability of the VOCs of the endophytic fungus *Muscodor crispans* isolated from *Ananas ananassoides* to inhibit the growth of phytopathogens, among which there were *Phytophthora cinnamomi* and *P. palmivora*. The VOCs produced by *M. crispans* that were composed mainly of propanoic acid, 2-methyl; propanoic acid, 2-methyl-; 1-butanol, 3-methyl-; 1-butanol, 3-methyl-, acetate; propanoic acid, 2-methyl-, 2-methylbutyl ester; and ethanol and were able to inhibit the growth of *Phytophthora cinnamomi* and *P. palmivora* by 100% with an IC50 (μL mL−1) of 0.056 and < 0.02, respectively. Mathew *et al.,* (2011) [57] isolated two endophytic fungi identified as *Trichoderma viride* and *T. pseudokoningii* from black pepper plants which showed *in vitro* inhibition capacity against *Phytophthora capsici* with an inhibition percentage of 64.4% and 65.6%, respectively. In the *in vivo* study, the lowest percentage in the incidence and severity of the disease caused by *P. capsici* was presented by the strain identified as *T. viride*. Bae *et al.*, (2011) [58] evaluated the antagonism capacity against *Phytophthora capsici* of six species of *Trichoderma* (*T. ovalisporum, T. theobromicola, T. hamatum, T. stilbohypoxyli, T. caribbaeum* var. *aequatoriale* and *T. theobromicola*) isolated from *Banisteriopsis caapi*, *Theobroma cacao*, *Theobroma gileri*, and *Cola praecuta*. All strains except for *T. caribbaeum* var. *aequatoriale* showed the ability to parasitize the mycelium of *P. capsici*. However, the culture filters of *T. caribbaeum* var. *aequatoriale* completely prevented growth of *P. capsici*, while *T. stilbohypoxyli* and *T. ovalisporum* presented inhibition percentages of 56.5% and 30.7, respectively. In addition, it was shown that the inoculation of *Trichoderma* strains in pepper seedlings activated genes associated with responsive to stress. *In vivo* tests, the strain identified as *T. theobromicola* delayed the onset of disease symptoms for more than 3 days and between 26 and 60% of the pepper seedlings remained asymptomatic. Miles *et al.,* (2012) [59] studied the biocontrol potential of 100 fungal endophytes isolated from *Espeletia* spp. Among the phytopathogens used to measure this potential was *Phytophthora infestans*. The growth of *P. infestans in vitro* was completely inhibited by eight endophytes which were identified as *Aureobasidium pullulans*, *Nigrospora oryzae*, *Chaetomium globosum*, *Trichoderma asperellum* and *Penicillium commune*. The crude extract of the culture of *A. pullulans* and *P. commune* also showed the ability to inhibit 100% the growth of *P. infestans*. Tellenbach *et al.,* (2013) [60] evaluated the ability of *Phialocephala europaea* isolated from *Picea abies* to inhibit the growth of *Phytophthora citricola* s.l. The strain of *P. europaea* was able to reduce the growth of *P. citricola in vitro*. The four compounds isolated from this microorganism were identified as sclerin, sclerolide, sclerotinin A and sclerotinin B. Sclerin and sclerotinin A were the main compounds produced, which *in vitro* significantly reduced the growth of *P. citrícola* at a concentration of 30 mg mL−1. Park *et al.,* (2015) [61] isolated the endophytic fungi identified as *Phoma terrestris*, *Fusarium oxysporum* and *Ascomycete* spp. from *Panax quinquefolius*, which inhibited the growth of *Phytophthora cactorum* with percentages between 64% to 82% and from 71% to 80% in the disk diffusion tests and fermentation broth tests, respectively. The main metabolites produced by *P. terrestris, F. oxysporum* and *Ascomycete* spp., were identified as N-amino-3-hydroxy-6-methoxyphthalimide, 3-methylthiobenzothiophene, phthalic acid, erucylamide and 2H-1-benzopyran-2-1, 3,4,5,6,7,8-hexahydro-4,7-dimethyl-. In the enzyme assays, the endophytic fungus identified as *P. terrestris* showed activity for the cellulase, xylanase, β-glucanase, pectinase and chitinase enzymes that could play a role in the inhibition of phytopathogens.

*Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

Terhonen *et al.,* (2016) [62] isolated the endophytic fungi identified as *Cryptosporiopsis* spp. and *Phialocephala sphareoides* from *Picea abies* which were able to inhibit the growth of *Phytophthora pini in vitro*. In addition, a decrease in the growth of *P. pini* was observed when the crude extract of the culture medium of *Cryptosporiopsis* spp. were tested. Subsequently, the analysis of the crude extract by UPLC-QTOF/MS was able to establish that the main metabolites produced by *Cryptosporiopsis* spp. had the following chemical formula C19H30O6, C20H28O8, C20H30O7 and C18H28O6. Sreeja *et al.,* (2016) [63] isolated 125 endophytic fungi from *Piper nigrum* which were evaluated to measure the ability to inhibit *Phytophthora capsici in vitro*. Of the 125 isolated fungi, 23 presented this capacity in more than 50%. The fungi with the highest inhibition capacity (78%) were identified as *Ceriporia lacerate*, *Phomopsis* spp. and *Diaporthe* spp. Other strains identified as *Daldinia eschscholtzii*, *Annulohypoxylon nitens* and *Fusarium* spp. presented inhibition capacity between 74% to 75%. Competition, VOCs antibiosis and mycoparasitism were reported to be among the biocontrol strategies for these fungi against *P. capsica*. Wang *et al.,* (2016) established by genome mining the biocontrol capacity of two strains of *Purpureocillium lilacinum* (PLBJ-1 and PLFJ-1) isolated from *Solanum lycopersicum*. Among the genes detected that may be useful in biocontrol were those that code for CAZymes, protease, glycoside hydrolases, and carbohydrate esterase. Regarding the production of secondary metabolites, genes coding for polyketide synthase, non-ribosomal peptide synthetase, terpene synthase and dimethylallyl tryptophan synthase were detected. Among these genes, those responsible for the synthesis of leucinostatin A and B were detected, which was confirmed by the production of mutants incapable of producing this compound. *In vitro* tests with the wild type and the mutant strain showed that the synthesis of leucinostatin A and B is closely related to the ability of these strains to inhibit the growth of *Phytophthora infestans* and *P. capsici*. Sanchez-Ortiz *et al.*, (2016) [65] evaluated the biocontrol capacity and VOCs of the endophytic fungus of *Haematoxylon brasiletto* Karst identified as *Xylaria* spp. PB3f3. The endophytic fungus was able to inhibit *Phytophthora capsici* by 48.3% *in vitro* and it was able to produce forty VOCs composed mainly of 3-methyl-1-butanol and thujopsene. Sánchez-Fernández *et al.* (2020) [66] studied antifungal and antioomycete activities of the compounds synthesized by the endophytic fungus *Hypoxylon anthochroum* isolated from *Gliricidia sepium*. The chemical study of the culture medium and the organic extracts of mycelium of the endophytic fungus led to the isolation of three isobenzofuranones: 7-hydroxy-4,6-dimethyl-3H-isobenzofuran-1-one **(1)**, 7-methoxy-4, 6-dimethyl-3H-isobenzofuran-1-one **(2)**, 6-formyl-4-methyl-7-methoxy-3H-isobenzofuran-1-one **(3)** and one compound was isolated for the first time as a natural product, 7- methoxy-4-methyl-3H-isobenzofuran-1-one **(4)** and another obtained by chemical synthesis 7-methoxy-6-methyl-3H-isobenzofuran-1-one **(5),** which showed the ability to inhibit the radial growth of *Phytophthora capsici* with an IC50 mM of 0.76, 0.62,> 0.97,> 1.12 and 2.12 respectively. Regarding the ability to alter the permeability of the *P. capsici* membrane, compounds 1, 2 and 5 presented an IC50 mM of <1.40, 0.55 and 2.03, respectively. In addition, these compounds were able to inhibit the respiration of *P. capsici*, being 2 the most efficient with an IC50 mM of 0.34.

#### **5. Conclusions**

Currently, the control of infections caused by *Phytophthora* spp. is very complicated, mainly due to the fact that many of the fungicides available on the market are not effective against this oomycete and also many of them are associated with

#### *Agro-Economic Risks of* Phytophthora *and an Effective Biocontrol Approach*

environmental and health damage. Therefore, the use of biocontrol agents as an alternative opens the possibility of using endophytic microorganisms, associated with the plant environment, which show great potential against this oomycete. Endophytic microorganisms isolated from different plant species have shown the ability to inhibit the growth of different *Phytophthora* species through various mechanisms such as antibiosis, VOCs, enzyme production, competition, among others. Therefore, the isolation of endophytic microorganisms and the study of their antagonistic capacity allows us to find new biocontrol agents, or their bioactive molecules, that allow controlling the enormous economic losses caused by *Phytophthora* spp.

### **Author details**

Hernando José Bolivar-Anillo1 , Victoria E. González-Rodríguez2 , Giovanna Reyes Almeida1 , Inmaculada Izquierdo-Bueno2 , Javier Moraga2 , María Carbú<sup>2</sup> , Jesús M. Cantoral<sup>2</sup> and Carlos Garrido2 \*

1 Programa de Microbiología, Facultad de Ciencias Básicas y Biomédicas, Universidad Simón Bolívar, Barranquilla, Colombia

2 Facultad de Ciencias del Mar y Ambientales, Laboratorio de Microbiología, Departamento de Biomedicina, Biotecnología y Salud Pública, Universidad de Cádiz, Puerto Real, España

\*Address all correspondence to: carlos.garrido@uca.es

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Endophytic Microorganisms as an Alternative for the Biocontrol of* Phytophthora *spp. DOI: http://dx.doi.org/10.5772/intechopen.99696*

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## Section 3
