Chapter 5 - Actinobacteria: Eco-Friendly Candidates for Control of Plant Diseases in a Sustainable Manner

ABSTRACTThe oomycete Pythium oligandrum is a potential biocontrol agent to control a wide range of fungal and oomycete-caused diseases, such as Pythium myriotylum-caused rhizome rot in ginger, leading to reduced yields and compromised quality. Previously, P. oligandrum has been studied for its plant growth-promoting potential by auxin production and induction of disease resistance by elicitors such as oligandrin. Volatile organic compounds (VOCs) play beneficial roles in sustainable agriculture by enhancing plant growth and resistance. We investigated the contribution of P. oligandrum-produced VOCs on plant growth and disease suppression by initially using Nicotiana benthamiana plants for screening. P. oligandrum VOCs significantly enhanced tobacco seedling and plant biomass contents. Screening of the individual VOCs showed that 3-octanone and hexadecane promoted the growth of tobacco seedlings. The total VOCs from P. oligandrum also enhanced the shoot and root growth of ginger plants. Transcriptomic analysis showed a higher expression of genes related to plant growth hormones and stress responses in the leaves of ginger plants exposed to P. oligandrum VOCs. The concentrations of plant growth hormones such as auxin, zeatin, and gibberellic acid were higher in the leaves of ginger plants exposed to P. oligandrum VOCs. In a ginger disease biocontrol assay, the VOC-exposed ginger plants infected with P. myriotylum had lower levels of disease severity. We conclude that this study contributes to understanding the growth-promoting mechanisms of P. oligandrum on ginger and tobacco, priming of ginger plants against various stresses, and the mechanisms of action of P. oligandrum as a biocontrol agent.IMPORTANCE Plant growth promotion plays a vital role in enhancing production of agricultural crops, and Pythium oligandrum is known for its plant growth-promoting potential through production of auxins and induction of resistance by elicitors. This study highlights the significance of P. oligandrum-produced VOCs in plant growth promotion and disease resistance. Transcriptomic analyses of leaves of ginger plants exposed to P. oligandrum VOCs revealed the upregulation of genes involved in plant growth hormone signaling and stress responses. Moreover, the concentration of growth hormones significantly increased in P. oligandrum VOC-exposed ginger plants. Additionally, the disease severity was reduced in P. myriotylum-infected ginger plants exposed to P. oligandrum VOCs. In ginger, P. myriotylum-caused rhizome rot disease results in severe losses, and biocontrol has a role as part of an integrated pest management strategy for rhizome rot disease. Overall, growth enhancement and disease reduction in plants exposed to P. oligandrum-produced VOCs contribute to its role as a biocontrol agent.

In the present study, 129 rhizospheric bacteria isolated from Curcuma longa were screened for their antagonistic potential against six fungal phytopathogens. Among them, 32 isolates that showed significant antagonistic potential were screened for their in vitro plant growth promoting (PGP) traits. The identification of potential isolates was confirmed by 16S rRNA gene sequencing and results revealed Bacillus as the dominant genus followed by Staphylococcus, Pseudomonas, Sphingomonas and Achromobacter. Based on the antagonistic activity and PGP traits; two strains (BPSRB4 and BPSRB14), identified as Bacillus amyloliquefaciens, were further tested for their in vivo PGP and disease suppression potential on Capsicum annuum seedlings under greenhouse conditions. The results demonstrated that BPSRB4 and BPSR14 strains suppress fungal pathogen infection and promote plant growth. Further, the BPSRB4 strain was positive for the production of the phytohormone indole acetic acid (IAA) detected by thin layer chromatography (TLC). In addition, nitrogen fixation and plant growth promotion activity were also confirmed by amplification and sequencing of nitrogen fixation gene (nifH) and ACC (1-aminocyclopropane-1-carboxylate) deaminase (acdS) gene from strains BPSRB4 and BPSRB14. The present study demonstrated that the B. amyloliquefaciens strains BPSRB4 and BPSR14 possess antagonistic activity and PGP potential which could be explored for the development of biofertilizers and biocontrol agents for the growth of chilli seedlings.

The northern root-knot nematode, Meloidogyne hapla, is a major pest of many crop species. The objective of the study was to determine how M. hapla population dynamics is affected by two precrops, i.e., Trifolium pratense and Medicago sativa, in three crop durations: one, two and three years of continuous cultivation. Moreover, we set ourselves the task of evaluating the effect of the legume precrop soil on the growth of the succeeding tomato plant (Solanum lycopersicum) and on the nematode population. The experiment was performed outdoors in pots with naturally infected soil. Both precrop species investigated were found to modify the J2 nematode population density in the soil. The galls and nematode females with egg masses were observed on the roots of both studied plant species at the end of each growing season. They appeared to be more abundant on the red clover roots than on those of the alfalfa. The obtained data indicate that the spring soil sampling is more appropriate for the estimation of the M. hapla population density in the red clover precrop soil. The legume precrop soil had a limiting effect on tomato growth and fruit yield. The nematode population negatively influenced tomato growth. The experiment revealed that tomato plants could be planted in alfalfa precrop soil following at least three years of continuous alfalfa cultivation. The same cannot be said of the cultivation of red clover as a precrop for tomatoes.

ilamentous fungi of the genus Colletotrichum and its teleomorph Glomerella are considered major plant pathogens worldwide. They cause significant economic damage to crops in tropical, subtropical, and temperate regions. Cereals, legumes, ornamentals, vegetables, and fruit trees may be seriously affected by the pathogen (3). Although many cultivated fruit crops are infected by Colletotrichum species, the most significant economic losses are incurred when the fruiting stage is attacked. Colletotrichum species cause typical disease symptoms known as anthracnose, characterized by sunken necrotic tissue where orange conidial masses are produced. Anthracnose diseases appear in both developing and mature plant tissues (4). Two distinct types of diseases occur: those affecting developing fruit in the field (preharvest) and those damaging mature fruit during storage (postharvest). The ability to cause latent or quiescent infections has grouped Colletotrichum among the most important postharvest pathogens. Species of the pathogen appear predominantly on aboveground plant tissues; however, belowground organs, such as roots and tubers, may also be affected. In this article, we deal in particular with methods used to identify and characterize Colletotrichum species and genotypes from almond, avocado, and strawberry, as examples, using traditional and molecular tools. The three pathosystems chosen represent different disease patterns of fruitassociated Colletotrichum. Multiple Species on a Single Host Numerous cases have been reported in which several Colletotrichum species or biotypes are associated with a single host. For example, avocado and mango anthracnose, caused by both C. acutatum and C. gloeosporioides, affect fruit predominantly as postharvest diseases (25,40,41). Strawberry may be infected by three Colletotrichum species, C. fragariae, C. acutatum, and C. gloeosporioides, causing anthracnose of fruit and other plant parts (31). Almond and other deciduous fruits may be infected by C. acutatum or C. gloeosporioides (Table 1) (1,5,46,50). Citrus can be affected by four different Colletotrichum diseases (61): postbloom fruit drop and key lime anthracnose, both caused by C. acutatum, and shoot dieback and leaf spot, and postharvest fruit decay, both caused by C. gloeosporioides. Additional examples of hosts affected by multiple Colletotrichum species include coffee, cucurbits, pepper, and tomato. Single Species on Multiple Hosts It is common to find that a single botanical species of Colletotrichum infects multiple hosts. For example, C. gloeosporioides (Penz.) Penz. & Sacc. in Penz. (teleomorph: Glomerella cingulata (Stoneman) Spauld. & H. Schrenk), which is considered a cumulative species and forms the sexual stage in some instances, is found on a wide variety of fruits, including almond, avocado, apple, and strawberry (Table 2) (6,15,31,46). Likewise, C. acutatum J.H. Simmonds has been reported to infect a large number of fruit crops, including avocado, strawberry, almond, apple, and peach (1,5,16,25,27). Examples of other species with multiple host ranges include C. coccodes, C. capsici, and C. dematium (14,56).

Suppression of Alternaria blight disease and plant growth promotion of mustard (Brassica juncea L.) by antagonistic rhizosphere bacteria

Chapter 10 - Plant growth promoting microbes: a future trend for environmental sustainability

Phytophthora blight, caused by the oomycete pathogen, Phytophthora capsici, is a devastating disease on bell pepper and cucurbit crops in the United States and worldwide (29,40). P. capsici causes a root and crown rot, as well as an aerial blight of leaves, fruit, and stems, on bell pepper (Capsicum annuum), tomatoes, cucumber, watermelon, squash, and pumpkin (29,35, 40,57,73). The disease was first described on bell pepper in New Mexico in 1922 (40). In recent years, epidemics have been severe in areas of North Carolina, Florida, Georgia, Michigan, and New Jersey. Oospores are believed to provide the initial source of inoculum in the field, and the disease is polycyclic within seasons (1,7,59,60,67). In this article, we discuss the biology and epidemiology of Phytophthora blight on bell pepper and also describe management strategies that can be implemented based on existing knowledge of the ecology of this devastating pathogen. The objectives of ecologically based pest management (EBPM) are the safe, profitable, and durable management of pests that includes a total systems approach (25). EBPM relies primarily on biological input of knowledge concerning a pathogen life cycle, and secondarily, when necessary, on physical, chemical, and biological supplements for disease management. An understanding of the ecological processes that are suppressive to plant diseases is emphasized rather than secondary inputs (25). Fortunately, we have a considerable amount of information available on the biology and ecology of P. capsici, which can now be integrated to improve our ability to manage the disease using ecologically based approaches. Strategies recommended for management of Phytophthora blight involve integrated approaches that focus first on cultural practices that reduce high soil moisture conditions, but also include monitoring and reduction of propagules of P. capsici that persist in the soil, utilization of cultivars with resistance to the disease, and when necessary, judicious fungicide applications. Symptoms and Life Cycle P. capsici can infect virtually every part of the pepper plant. The pathogen causes a root and crown rot on pepper (Fig. 1) and also forms distinctive black lesions on the stem (Fig. 2). P. capsici can also infect the leaves and causes lesions that are circular, grayish brown, and water-soaked (Fig. 3). Leaf lesions and stem lesions are common when inoculum is splash dispersed from the soil to lower portions of the plant. The pathogen can also infect fruit and causes lesions that are typically covered with white sporangia, a sign of the pathogen (Fig. 4). P. capsici typically causes a fruit rot or stem rot on cucumbers and squash (Fig. 5). P. capsici reproduces by both sexual and asexual means (Fig. 6). The pathogen produces two mating types, known as the A1 and A2. These are actually compatibility types and do not correspond to dimorphic forms. Each mating type produces hormones that are responsible for gametangia differentiation in the opposite mating type. Both A1 and A2 mating types are common in fields in North Carolina and have also been identified within the same plant (59). P. capsici produces a male gametangium, called the antheridium, and a female gametangium, called the oogonium. The antheridium is amphigynous in this species. Meiosis occurs within the gametangia, and plasmogamy and karyogamy result

Effective use of biocontrol agents is an important component of sustainable agriculture. A previous numerical study of a generic model showed that biocontrol efficacy was greatest for a single biocontrol agent (BCA) combining competition with mycoparasitism or antibiosis. This study uses the same mathematical model to investigate whether the biocontrol efficacy of combined use of two BCAs with different biocontrol mechanisms is greater than that of a single BCA with either or both of the two mechanisms, assuming that two BCAs occupy the same host tissue as the pathogen. Within the parameter values considered, a BCA with two biocontrol mechanisms always outperformed the combined use of two BCAs with a single but different biocontrol mechanism. Similarly, combined use of two BCAs with a single but different biocontrol mechanism is shown to be far less effective than that of a single BCA with both mechanisms. Disease suppression from combined use of two BCAs was very similar to that achieved by the more efficacious one. As expected, a higher BCA introduction rate led to increased disease suppression. Incorporation of interactions between two BCAs did not greatly affect the disease dynamics except when a mycoparasitic and, to a lesser extent, an antibiotic-producing BCA was involved. Increasing the competitiveness of a mycoparasitic BCA over a BCA whose biocontrol mechanism is either competition or antibiosis may lead to improved biocontrol initially and reduced fluctuations in disease dynamics. The present study suggests that, under the model assumptions, combined use of two BCAs with different biocontrol mechanisms in most cases only results in control efficacies similar to using the more efficacious one alone. These predictions are consistent with published experimental results, suggesting that combined use of BCAs should not be recommended without clear understanding of their main biocontrol mechanisms and relative competitiveness, and experimental evaluation.

Chapter 25 - Exploring the potential role of Trichoderma as friends of plants foes for bacterial plant pathogens

Diversity and biotechnological potential of endophytic Bacillus species originating from the stem apoplast fluid of sugarcane plants

Deleterious effects of artificially applied chemicals have highlighted the significance of biocontrol agents as suitable substitute for sustainable agriculture. In present study, three endophytic bacterial strains SV7, SV10 and LV19 showed extensive range of antifungal as well as plant growth promoting activities signifying potential to accomplish the requirement. Phylogenetic analysis revealed 100% similarity of three strains with taxon Firmicute. However, there was division among these isolates on basis of subgroups as SV7 belonged to Exiguobacterium auranticum, SV10 belonged to Paenibacillus sp. and LV19 was best fit in subgroup Priestia koreensis. All strains showed antifungal activity against Fusarium oxysporum on three different media (PDA, NA, LA) with maximum activity (53%) of LV19 strain on NA and least activity (13%) on PDA medium as recorded by zones of inhibition. In growth promotion experiments, combination of LV19 with Fusarium significantly suppressed chances of Fusarium wilt which is commonly caused by Fusarium oxysporum in sunflower plants. Diverse growth parameters (seed germination percentage, lengths and fresh weights of root and shoot) were significantly increased from 34 to 909% over pathogen infected plants only which was further proved by their root colonization analysis. Based on most efficient growth promotion by LV19 strain, expression of five plant defense related genes (SOD, PAL, NPR1, PR5, Chitinase) was evaluated revealing enhanced expression by 1.7-270-folds in consortium of LV19 and Fusarium. Thus, current study provided a scientific justification that bacterial strains in specific LV19 (Priestia koreensis) could be further developed as biocontrol agent with potential of plant growth promotion.

The exploitation of the association between plants and microorganisms is a promising approach able to boost natural attenuation processes for soil clean-up in vast polluted areas characterized by mixed chemical contamination. We aimed to explore the selection of root-associated bacterial communities driven by different plant species spontaneously established in abandoned agricultural soils within a historical polluted site in north Italy. The site is highly contaminated by chlorinated persistent organic pollutants, mainly constituted by polychlorobiphenyls (PCBs), together with heavy metals and metalloids, in variable concentrations and uneven distribution. The overall structure of the non-vegetated and root-associated soil fractions bacterial communities was described by high-throughput sequencing of the 16S rRNA gene, and a collection of 165 rhizobacterial isolates able to use biphenyl as unique carbon source was assayed for plant growth promotion (PGP) traits and bioremediation potential. The results showed that the recruitment of specific bacterial communities in the root-associated soil fractions was driven by both soil fractions and plant species, explaining 21 and 18% of the total bacterial microbiome variation, respectively. PCR-based detection in the soil metagenome of bacterial bphA gene, encoding for the biphenyl dioxygenase α subunit, indicated that the soil in the site possesses metabolic traits linked to PCB degradation. Biphenyl-utilizing bacteria isolated from the rhizosphere of the three different plant species showed low phylogenetic diversity and well represented functional traits, in terms of PGP and bioremediation potential. On average, 72% of the strains harbored the bphA gene and/or displayed catechol 2,3-dioxygenase activity, involved in aromatic ring cleavage. PGP traits, including 1-aminocyclopropane-1-carboxylic acid deaminase activity potentially associated to plant stress tolerance induction, were widely distributed among the isolates according to in vitro assays. PGP tested in vivo on tomato plants using eleven selected bacterial isolates, confirmed the promotion and protection potential of the rhizosphere bacteria. Different spontaneous plant species naturally selected in a historical chronically polluted site showed to determine the enrichment of peculiar bacterial communities in the soil fractions associated to the roots. All the rhizosphere communities, nevertheless, hosted bacteria with degradation/detoxification and PGP potential, putatively sustaining the natural attenuation process.

Chapter 11 - Plant growth promoting bacteria as biocontrol agents against diseases of cereal crops

Food production must undergo systems change to meet the sustainable development goals (SDGs). For example, organic farming can be empowered by soil microorganisms with plant growth promotion (PGP) and biocontrol features. In this context, there have been limited studies on pomegranate. We investigated microbial diversity in rhizosphere of the pomegranate "Bhagwa" variety and its potential role in PGP and biocontrol. Both bulk and rhizosphere soil samples were analyzed for their physicochemical properties. Whole metagenome sequencing was conducted using the Illumina NovaSeq6000 platform. Surprisingly, we found that bulk and rhizosphere soil samples had comparable microbial diversity. Metagenome sequencing revealed the abundance of Streptomyces indicus, Bradyrhizobium kalamazoonesis, and Pseudomonas cellulosum in the rhizosphere that are reported here for the first time in agricultural literature. Pathway prediction analysis using KEGG (Kyoto Encyclopedia for Genes and Genomes) and COG (clusters of orthologous genes) databases identified metabolic pathways associated with biocontrol properties against pathogens. We confirmed the metagenome data in vitro, which demonstrated their PGP potential and antimicrobial properties. For instance, S. indicus produced high concentration of indole-3-acetic acid, a PGP phytohormone, that can stimulate plant growth. In addition, an antimicrobial susceptibility assay suggested that bacterial extracts displayed activity against Xanthomonas, a primary pathogen causing the pomegranate wilt disease. In conclusion, this study suggests that S. indicus, B. kalamazoonesis, and P. cellulosum can potentially be PGP and biocontrol agents that may contribute to increased crop productivity in pomegranate cultivation. These agents and their combinations warrant future research with an eye on SDGs and so as to enable and innovate organic farming and pomegranate agricultural practices.

This study evaluated the phylogenetic diversity, plant growth promotion capacity, antifungal activity, and biocontrol potential of culturable actinobacterial endophytes isolated from the medicinal plant Aconitum carmichaelii Debeaux. Isolation of actinobacteria from healthy A. carmichaelii plants was carried out on six different media. Full-length 16S rRNA gene was amplified by PCR from the genomic DNA of each strain. Indole-3-acetic acid and siderophore production were quantitatively assessed by the Salkowski and Chrome Azurol S methods, respectively. Rice seeds germination and seedling growth were employed to evaluate plant growth promotion capacities of candidate strains. Dual-culture assay and pot experiments were performed to investigate the antifungal and biocontrol potential of isolates. We obtained 129 actinobacterial isolates from A. carmichaelii, and they belonged to 49 species in 7 genera. These strains exhibited diverse plant growth promotion ability, among which one strain significantly enhanced rice seeds germination, while 31 strains significantly facilitated rice seedling growth. SWUST-123 showed strong antifungal activity against four pathogens in vitro and was most compatible with Qingchuan cultivar. SWUST-123 reduced around 40% of southern blight disease occurrence compared to blank control treatment. . Aconitum carmichaelii harbored genetically diverse actinobacterial endophytes exhibiting diverse plant growth promotion and antifungal potential, some of which can be served as good candidates for biofertilizers and biocontrol agents.