Endophytic Metarhizium anisopliae isolates suppress two-spotted spider mite population and black leaf spot disease while enhancing tomato plant growth

Bamisile B.S., Afolabi O.G., Siddiqui J.A., Xu Y.

Various techniques used by crop plants to evade insect pests and pathogen attacks have been documented. Among these, plant defense strategies induced by endophytic insect pathogenic fungi are arguably one of the most discussed. Endophytic fungi frequently colonize plants and inhabit their internal tissues for a portion of their lifespan without producing visible symptoms of the disease. This phenomenon is widespread and diverse in both natural and agricultural ecosystems, and is present in almost all plant organs. Many fungi can obtain nutrients by infecting and killing insects, and this ability has been developed numerous times in different fungal lineages. These species mainly consist of those in the order Hypocreales (Ascomycota), where the generalist insect pathogens, Beauveria sp. (Cordycipitaceae) and Metarhizium sp. (Clavicipitaceae) are two of the most studied endophytic entomopathogenic fungal genera. However, most fungi that kill insects do not survive in the tissues of living plants. The data published thus far show a high degree of variability and do not provide consistent explanations for the underlying mechanisms that may be responsible for these effects. This implies that available knowledge regarding the colonization of plant tissues by endophytic insect pathogenic fungi, the effects of colonization on plant metabolism, and how this contributes to a decrease in herbivore and pathogens damage is limited. To adequately utilize fungal-based products as biological control agents, these products must be effective and the reduction of pests and infection must be consistent and similar to that of chemical insecticides after application. This article discusses this possibility and highlights the benefits and the specific techniques utilized by endophytically challenged plants in invading insect pests and disease pathogens.

Sallam N.M., AbdElfatah H.S., Khalil Bagy H.M., Elfarash A., Abo-Elyousr K.A., Sikora E.J., Sallam A.

Controlling early blight of tomatoes using endophytic bacteria is an eco-friendly and sustainable approach to manage this common fungal disease caused by Alternaria solani, Alternaria alternata, and Curvularia lunata. Endophytic bacteria are microorganisms that live inside plant tissues without causing harm and can help protect the host plant from pathogens. In this work, twenty endophytic bacterial isolates from tomato healthy plants were tested against pathogenic fungal isolates that caused early blight disease in vitro. Out of the 20 tested isolates, three (B4, B7, and B17) were considered effective isolates against the growth of fungal pathogens. The three isolates were recognized as Enterobacter cloacae HS-6 (B4), Pseudomonas gessardii HS-5 (B 7), and Pseudomonas mediterranea HS-4 (B17) using 16s-rDNA sequencing. Different concentrations of bacterial cultural diltrates at 20, 40, and 60% were tested for their antagonistic effects on the development of pathogenic fungi in vitro. The lowest dry weights of pathogenic isolates in all bacterial culture filtrates were discovered at 60%. In all culture filtrates, phenolic compounds showed the largest peak area. Under greenhouse conditions, the least disease severity of tomato early blight was found for E. cloacae and its culture filtrate compared to other treatments. Real-time PCR was used to examine the expression pattern of the defense response gene β-1.3 glucanase gene in infected tomato plants with pathogenic fungi (control) as well as its relations with efficient biocontrol agent (E. cloacae). The expression of the gene increased substantially and significantly after three days from the inoculation-infected plants with C. lunata and E. cloacae while it reached the maximum after five days from the inoculation with A. alternata, A. solani and E. cloacae. Our study concluded that the endophytic bacterial isolate E. cloacae can be considered a promising biocontrol agent for preventing tomato early blight.

Mimma A.A., Akter T., Haque M.A., Baki Bhuiyan M.A., Hasan Chowdhury M.Z., Sultana S., Naimul Islam S.M.

Rhizoctonia solani is an important necrotrophic pathogenic fungus that causes okra root disease and results in severe yield reduction. Many biocontrol agents are being studied with the intent of improving plant growth and defense systems and reducing crop loss by preventing fungal infections. Recently, a member of the Hypocrealean family, Metarhizium anisopliae, has been reported for insect pathogenicity, endophytism, plant growth promotion, and antifungal potentialities. This research investigated the role of M. anisopliae (MetA1) in growth promotion and root disease suppression in okra. The antagonism against R. solani and the plant growth promotion traits of MetA1 were tested in vitro. The effects of endophytic MetA1 on promoting plant growth and disease suppression were assessed in planta. Dual culture and cell-free culture filtrate assays showed antagonistic activity against R. solani by MetA1. Some plant growth promotion traits, such as phosphate solubilization and catalase activity were also exhibited by MetA1. Seed primed with MetA1 increased the shoot, root, leaves, chlorophyll content, and biomass content compared to control okra plants. The plants challenged with R. solani showed the highest hydrogen peroxide (H2O2) and lipid peroxidation (MDA) contents in the leaves of okra. Whereas MetA1 applied plants showed a reduction of H2O2 and MDA by 5.21 and 14.96%, respectively, under pathogen-inoculated conditions by increasing antioxidant enzyme activities, including catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), and ascorbate peroxidase (APX), by 30.11, 10.19, 5.62, and 5.06%, respectively. Moreover, MetA1 increased soluble sugars, carbohydrates, proline, and secondary metabolites, viz., phenol and flavonoid contents in okra resulting in a better osmotic adjustment of diseases infecting plants. MetA1 reduced disease incidence by 58.33% at 15 DAI compared to the R. solani inoculated plant. The results revealed that MetA1 improved plant growth, elevated the plant defense system, and suppressed root diseases caused by R. solani. Thus, MetA1 was found to be an effective candidate for the biological control program.

Posada-Vergara C., Vidal S., Rostás M.

Metarhizium brunneum is a soil-borne fungal entomopathogen that can be associated with plant roots. Previous studies have demonstrated that root colonization by beneficial fungi can directly affect soil-borne pathogens through competition and antibiosis and can activate a systemic response in plants, resulting in a primed state for a faster and/or stronger response to stressors. However, the mechanisms by which Metarhizium inoculation ameliorates symptoms caused by plant pathogens are not well known. This study evaluated the ability of M. brunneum to protect oilseed rape (Brassica napus L.) plants against the soil-borne pathogen Verticillium longisporum and investigated whether the observed effects are a result of direct interaction and/or plant-mediated effects. In vitro and greenhouse experiments were conducted to measure fungal colonization of the rhizosphere and plant tissues, and targeted gene expression analysis was used to evaluate the plant response. The results show that M. brunneum delayed pathogen colonization of plant root tissues, resulting in decreased disease symptoms. Direct competition and antibiosis were found to be part of the mechanisms, as M. brunneum growth was stimulated by the pathogen and inhibited the in vitro growth of V. longisporum. Additionally, M. brunneum changed the plant response to the pathogen by locally activating key defense hormones in the salicylic acid (SA) and abscisic acid (ABA) pathways. Using a split-root setup, it was demonstrated that there is a plant-mediated effect, as improved plant growth and decreased disease symptoms were observed when M. brunneum was in the systemic compartment. Moreover, a stronger systemic induction of the gene PR1 suggested a priming effect, involving the SA pathway. Overall, this study sheds light on the mechanisms underlying the protective effects of M. brunneum against soil-borne pathogens in oilseed rape plants, highlighting the potential of this fungal entomopathogen as a biocontrol agent in sustainable agriculture.

Díaz-Urbano M., Goicoechea N., Velasco P., Poveda J.

A new more sustainable agricultural system needs to be developed to increase production without compromising human and animal health and preserving essential resources, such as soil, water and diversity. Bio-inoculants can be a tool to favor this transition, as they can replace or complement agrochemicals that do not meet the above premises. Bio-inoculants generated from endophytic filamentous fungi and mycorrhizal fungi, whether used individually, in combination with each other or with other microorganisms, stand out for their potential. This review provides information on how bio-inoculants based on these microorganisms have been shown to increase crop yield and quality through strategies, such as increasing nutrient uptake or levels of certain phytohormones. On how they can promote tolerance to abiotic stresses, including heavy metals, elevated temperatures, salinity or drought, through strategies, such as the accumulation of osmoregulatory substances or increasing the plant's root surface, among others. And finally, in the ability to protect the plant against pathogens and pests, either by inducing defense systems, competing for space or synthesizing metabolites with antibiotic activity. It should be noted that, although there are already commercial products using these microorganisms for agricultural purposes, such as biological control agents or biostimulants, it is expected that a deeper understanding of the mechanisms of action of the microorganisms, together with improved technical production processes, will lead to more effective, safer and cheaper products.

Golzan S.R., Talaei-Hassanloui R., Homayoonzadeh M., Safavi S.A.

Entomopathogenic fungi (EPF) are important biological control agents in pest management programs in agroecosystems against insect pests. EPF such as Beauveria bassiana (Bals.) Vuillemin and Metarhizium anisopliae (Metchn.) Sorokin produce a wide range of extracellular enzymes involved in disturbance of the first barrier in the insect cuticle comprising proteins, chitin, and lipids. Realizing relationships between the expression of these enzymes and fungal virulence might aid in development of effective mycoinsecticides. The virulence of B. bassiana (isolates TV and OZ1) and M. anisopliae (isolate CS1) were investigated on Plodia interpunctella (Hübner) larvae in this study. The third instar larvae were immersed in a suspension containing 1 × 108 conidia mL−1 of fungal conidial inoculum. The results revealed that all three fungal isolates caused mortality in larvae, but there was a considerable variation in their virulence. Total proteinase, exochitinase and lipase assays were done for these isolates. The TV isolate with the highest mortality with 41.7%, had the highest level of specific activity of exochitinase, protease, and lipase with 0.148, 0.654, and 0.190 U. mg−1 protein, respectively. In the current study, a positive correlation was determined between the virulence of fungal isolates and the activities of protease and lipase, but this link was not significant for exochitinase. Our results demonstrated that extracellular enzymes, particularly protease and lipase, may play a crucial role in the virulence of these fungal isolates against P. interpunctella larvae.

Altaf N., Ullah M.I., Afzal M., Arshad M., Ali S., Rizwan M., Al-Shuraym L.A., Alhelaify S.S., Sayed S.

The fall armyworm, Spodoptera frugiperda (Noctuidae; Lepidoptera), is a serious threat to food security as it has the potential to feed on over 353 plant species. To control this insect pest, endophytic colonization of entomopathogenic fungi (EPF) in plants is being considered as a safer and more effective alternative. This study evaluated the efficacy of two EPFs, Beauveria bassiana and Metarhizium anisopliae, for endophytic colonization using foliar spray and seed treatment methods on maize plants, and their impact on the survival, development, and fecundity of S. frugiperda. Both EPF effectively colonized the maize plants with foliar spray and seed treatment methods, resulting in 72–80% and 50–60% colonization rates, respectively, 14 days after inoculation. The EPF negatively impacted the development and fecundity of S. frugiperda. Larvae feeding on EPF-inoculated leaves had slower development (21.21 d for M. anisopliae and 20.64 d for B. bassiana) than the control treatment (20.27 d). The fecundity rate was also significantly reduced to 260.0–290.1 eggs/female with both EPF applications compared with the control treatment (435.6 eggs/female). Age-stage-specific parameters showed lower fecundity, life expectancy, and survival of S. frugiperda when they fed on both EPF-inoculated leaves compared with untreated leaves. Furthermore, both EPFs had a significant effect on population parameters such as intrinsic (r = 0.127 d−1 for B. bassiana, and r = 0.125 d−1 for M. anisopliae) and finite rate (λ = 1.135 d−1 for B. bassiana, and λ = 1.1333 d−1 for M. anisopliae) of S. frugiperda compared with the control (r = 0.133 d−1 and λ = 1.146 d−1). These findings suggest that EPF can be effectively used for the endophytic colonization of maize plants to control S. frugiperda. Therefore, these EPFs should be integrated into pest management programs for this pest.

Soliman S.A., Abdelhameed R.E., Metwally R.A.

AbstractAlternaria alternata that threatens pepper production and causes major economic harm is responsible for the leaf spot/blight disease. Chemical fungicides have been widely employed; unfortunately, fungicidal resistance is a current concern. Therefore, finding new environmentally friendly biocontrol agents is a future challenge. One of these friendly solutions is the use of bacterial endophytes that have been identified as a source of bioactive compounds. The current study investigates the in vivo and in vitro fungicidal potential of Bacillus amyloliquefaciens RaSh1 (MZ945930) against pathogenic A. alternata. In vitro, the results revealed that RaSh1 exhibited strong antagonistic activity against A. alternata. In addition to this, we inoculated pepper (Capsicum annuum L.) plants with B. amyloliquefaciens RaSh1 and infected them with A. alternata. As a result of A. alternata infection, which generated the highest leaf spot disease incidence (DI), the plant's growth indices and physio-biochemical characteristics significantly decreased, according to our findings. Our results also showed the abnormal and deformed cell structure using light and electron microscopy of A. alternata-infected leaves compared with other treatments. However, DI was greatly reduced with B. amyloliquefaciens RaSh1 application (40%) compared to pepper plants infected with A. alternata (80%), and this led to the largest increases in all identified physio-biochemical parameters, including the activity of the defense-related enzymes. Moreover, inoculation of pepper plants with B. amyloliquefaciens RaSh1 decreased electrolyte leakage by 19.53% and MDA content by 38.60% as compared to A. alternata infected ones. Our results show that the endophyte B. amyloliquefaciens RaSh1 has excellent potential as a biocontrol agent and positively affects pepper plant growth.

Shaalan R., Ibrahim L., As-sadi F., El Kayal W.

In natural systems, plant–endophyte interactions are important for reducing abiotic and biotic stresses in plants by producing a variety of metabolites that protect plants from pathogens and herbivores. Biocontrol strategies are increasingly being used as a viable alternative to chemical pesticides. Entomopathogenic fungi (EPF) are one of them, and they have been touted as a successful method for biological pest control in plants. Because EPF strains are sensitive to environmental conditions when sprayed, the recently discovered endophytic behavior of several EPF strains has improved their management. Cucumber mosaic virus (CMV) is one of the most common and serious plant viruses worldwide, infecting over 1200 plant species and being spread by more than 80 aphid species. CMV control is directed towards the use of chemical insecticides to eradicate its insect vectors. Endophytic EPF is currently being studied to control plant virus infection, and antagonistic effects have been reported. Metabolomics is an emerging research field for plant metabolite profiling and is employed to study plant–endophyte interactions. In the present research, metabolomics approaches were conducted to gain information into mechanisms involved in defense against CMV in endophytes Beauveria bassiana and Metarhizium anisopliae (EPF)-treated diseased cucumber plants. In addition, CMV-induced metabolic changes in cucumber plants were investigated. Our analysis indicated large differences in cucumber metabolites due to endophytes application. In total, six hundred and thirty-one metabolites were differentially expressed in endophyte-treated CMV diseased cucumber plants. Regulation of different kinds of amino acids, organic acids, and phenylpropanoids metabolites could provide insight about plant defense mechanism against CMV pathogen. Important metabolites were found to be regulated in diseased cucumber plants due to fungal endophytes treatment that could possibly confer tolerance to CMV disease.

Liu Y., Yang Y., Wang B.

Beauveria bassiana and Metarhizium anisopliae are two of the most important and widely used entomopathogenic fungi (EPFs) to control insect pests. Recent studies have revealed their function in promoting plant growth after artificial inoculation. To better assess fungal colonization and growth-promoting effects of B. bassiana and M. anisopliae on crops, maize Zea mays seedlings were treated separately with 13 B. bassiana and 73 M. anisopliae as rhizosphere fungi in a hydroponic cultural system. Plant growth indexes, including plant height, root length, fresh weight, etc., were traced recorded for 35 days to prove the growth promoting efficiency of the EPFs inoculation. Fungal recovery rate (FRR) verified that both B. bassiana and M. anisopliae could endophytically colonize in maize tissues. The recovery rates of B. bassiana in stems and leaves were 100% on the 7th day, but dropped to 11.1% in the stems and 22.2% in the leaves on the 28th day. Meanwhile, B. bassiana was not detected in the roots until the 28th day, reaching a recovery rate of 33.3%. M. anisopliae strains were isolated from the plant roots, stems and leaves throughout the tracing period with high recovery rates. The systematical colonization of B. bassiana and M. anisopliae in different tissues were further corroborated by PCR amplification of fungus-specified DNA band, which showed a higher detection sensitivity of 100% positive reaction. Fungal density comparing to the initial value in the hydroponic solution, dropped to be well below 1% on the 21st day. Thus, the two selected entomopathogenic fungal strains successfully established endophytic colonization rather than rhizospheric colonization in maize, and significantly promoted its growth in a hydroponic cultural system. Entomopathogenic fungi have great application potential in eco-agricultural fields including biopesticides and biofertilizers.

Caccavo V., Forlano P., Mang S., Fanti P., Nuzzaci M., Battaglia D., Trotta V.

Fungi belonging to the genus Trichoderma have received much attention in recent years due to their beneficial effects on crop health and their use as pest control agents. Trichoderma activates direct plant defenses against phytophagous arthropods and reinforces indirect plant defense through the attraction of predators. Although the plant defenses against insect herbivores were demonstrated in laboratory experiments, little attention has been paid to the use of Trichoderma spp. in open field conditions. In the present study, we investigated the effects of the inoculation of the commercial Trichoderma harzianum strain T22 on the arthropod community associated with tomato plants and on the crop performance in an experimental field located in South Italy. Our results showed that inoculation with T. harzianum could alter the arthropod community and reduce the abundance of specific pests under field conditions with respect to the sampling period. The present study also confirmed the beneficial effect of T. harzianum against plant pathogens and on tomato fruit. The complex tomato–arthropod–microorganism interactions that occurred in the field are discussed to enrich our current information on the possibilities of using Trichoderma as a green alternative agent in agriculture.

González-Pérez E., Ortega-Amaro M.A., Bautista E., Delgado-Sánchez P., Jiménez-Bremont J.F.

Species of the entomopathogenic fungi Metarhizium are used worldwide as biocontrol agents. Recently, other lifestyles have been associated with some Metarhizium species, which include their role as saprophytes, endophytes, and plant growth promoters. Herein, the effect of three Metarhizium anisopliae strains on the growth of Arabidopsis thaliana plantlets was evaluated using an in vitro split system. Arabidopsis fresh weight and total chlorophyll content significantly increased 7 days post-inoculation with the three Metarhizium anisopliae strains evaluated. The primary root length was promoted by all fungal strains without physical contact, whereas in direct contact primary root growth was inhibited. Volatile organic compounds identification revealed that during the interaction of Arabidopsis with Ma-20 and Ma-25 strains only β-caryophyllene was produced, whereas in the Arabidopsis-Ma-28 interaction o-cymene was mainly emitted. The plant growth promoting effect induced by Metarhizium anisopliae strains was also achieved in Arabidopsis, tomato and maize plants grown in soil pots. Our results showed that three Metarhizium anisopliae strains were able to increase plant fresh weight, opening promising perspectives for field production, with the advantages of insect biocontrol and plant growth promotion induced by this species of fungus.

HOMAYOONZADEH M., ESMAEILY M., TALEBI K., ALLAHYARI H., REITZ S., MICHAUD J.P.

Al Khoury C.

Endophytic growth of arthropod pathogenic fungi can parasitize insect herbivores without causing damage to the crop. However, studies addressing this tritrophic interaction are absent. Here, the endophytic arthropod pathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordyciptaceae), the polyphagous two-spotted spider mite Tetranychus urticae Koch (Trombidiformes: Tetranychidae), and its preferred plant host Phaseolus vulgaris L. (Fabales: Fabaceae) were selected to study the multi-kingdom interactions among plants, arthropods, and entomopathogenic fungi. Real-Time PCR analysis of nine defense-related genes revealed that a broad range of plant defense mechanisms is activated in response to the endophytic growth of B. bassiana. Moreover, we studied the molecular mechanism adapted by the two-spotted spider mite that underlies resistance. The analysis of 41 detoxification genes revealed that relatively moderate, high, and few numbers of genes were changed in the adults, nymphs, and eggs stages of T. urticae, respectively, after inoculation on colonized tissues of P. vulgaris. The endophytic growth of B. bassiana can have a negative effect on the growth and performance of the pest, in a developmental stage-dependent manner, by priming plant defense pathways. In parallel, the herbivore induces a broad range of detoxification genes that could potentially be involved in adaptation to endophytically colonized plant tissues.