W. G. Dilantha Fernando

Molecular and biochemical detection of fengycin- and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of canola and wheat

Bacillus species are well known for their ability to control plant diseases through various mechanisms, including the production of secondary metabolites. Bacillus subtilis DFH08, an antagonist of Fusarium graminearum, and other Bacillus spp. that are antagonists of common fungal pathogens of canola were screened for peptide synthetase biosynthetic genes of fengycin and bacillomycin D. Specific polymerase chain reaction (PCR) primers identified B. subtilis strains DFH08 and 49 for the presence of the fenD gene of the fengycin operon. Bacillus cereus DFE4, Bacillus amyloliquefaciens strains DFE16 and BS6, and B. subtilis 49 were identified for the presence of the bamC gene of the bacillomycin D synthetase biosynthetic operon. Both fengycin and bacillomycin D were detected in the culture extract of strain Bs49, characterized through MALDI-TOF-MS (matrix-assisted laser desorption ionization - time of flight - mass spectrometry), and their antifungal activities demonstrated against F. graminearum and Sclerotinia sclerotiorum. This study designed and used specific PCR primers for the detection of potential fengycin- and bacillomycin D-producing bacterial antagonists and confirmed the molecular detection with the biochemical detection of the corresponding antibiotic produced. This is also the first report of a B. cereus strain (DFE4) to have bacillomycin D biosynthetic genes. Bacteria that synthesize these lipopeptides could act as natural genetic sources for genetic engineering of the peptide synthetases for production of novel peptides.

Pyrosequencing Reveals the Influence of Organic and Conventional Farming Systems on Bacterial Communities

It has been debated how different farming systems influence the composition of soil bacterial communities, which are crucial for maintaining soil health. In this research, we applied high-throughput pyrosequencing of V1 to V3 regions of bacterial 16S rRNA genes to gain further insight into how organic and conventional farming systems and crop rotation influence bulk soil bacterial communities. A 2×2 factorial experiment consisted of two agriculture management systems (organic versus conventional) and two crop rotations (flax-oat-fababean-wheat versus flax-alfalfa-alfalfa-wheat) was conducted at the Glenlea Long-Term Crop Rotation and Management Station, which is Canada’s oldest organic-conventional management study field. Results revealed that there is a significant difference in the composition of bacterial genera between organic and conventional management systems but crop rotation was not a discriminator factor. Organic farming was associated with higher relative abundance of Proteobacteria, while Actinobacteria and Chloroflexi were more abundant in conventional farming. The dominant genera including Blastococcus, Microlunatus, Pseudonocardia, Solirubrobacter, Brevundimonas, Pseudomonas, and Stenotrophomonas exhibited significant variation between the organic and conventional farming systems. The relative abundance of bacterial communities at the phylum and class level was correlated to soil pH rather than other edaphic properties. In addition, it was found that Proteobacteria and Actinobacteria were more sensitive to pH variation.

Plant Growth Promoting Rhizobacteria Formulations and its Scope in Commercialization for the Management of Pests and Diseases

The export oriented agricultural and horticultural crops depends on the export of residue free produce and has created a great potential and demand for the incorporation of biopesticides in crop protection. To ensure the sustained availability of biocontrol agent’s mass production technique and formulation development protocols has to be standardized to increase the shelf life of the formulation. It facilitates the industries to involve in commercial production of plant growth promoting rhizobacteria (PGPR). PGPR with wide scope for commercialization includes Pseudomonas fluorescens, P. putida, P. aeruginosa, Bacillus subtilis and other Bacillus spp. The potential PGPR isolates are formulated using different organic and inorganic carriers either through solid or liquid fermentation technologies. They are delivered either through seed treatment, bio-priming, seedling dip, soil application, foliar spray, fruit spray, hive insert, sucker treatment and sett treatment. Application of PGPR formulations with strain mixtures perform better than individual strains for the management of pest and diseases of crop plants, in addition to plant growth promotion. Supplementation of chitin in the formulation increases the efficacy of antagonists. More than 33 products of PGPR have been registered for commercial use in greenhouse and field in North America. Though PGPR has a potential scope in commercialization, the threat of certain PGPR (P. aeruginosa, P. cepacia and B. cereus) to infect human beings as opportunistic pathogens has to be clarified before large scale acceptance, registration and adoption of PGPR for pest and disease management.

BIOSYNTHESIS OF ANTIBIOTICS BY PGPR AND ITS RELATION IN BIOCONTROL OF PLANT DISEASES

Plant growth promoting rhizobacteria (PGPR) play a vital role in crop protection, growth promotion and in the improvement of soil health. Some well known PGPR strains are Pseudomonas, Bacillus, Azospirillum, Rhizobium, and Serratia species. The primary mechanism of biocontrol by PGPR involves the production of antibiotics such as phenazine-1-carboxyclic acid, 2,4-diacetyl phloroglucinol, oomycin, pyoluteorin, pyrrolnitrin, kanosamine, zwittermycin-A, and pantocin. A cascade of endogenous signals such as sensor kinases, N-acyl homoserine lactones and sigma factors regulates the synthesis of antibiotics. The genes responsible for the synthesis of antibiotics are highly conserved. The antibiotics pertain to polyketides, heterocyclic nitrogenous compounds and lipopeptides have broad-spectrum action against several plant pathogens, affecting crop plants. In addition to direct antipathogenic action, they also serve as determinants in triggering induced systemic resistance (ISR) in the plant system. Though antibiotics play a vital role in disease management, their role in biocontrol is questioned due to constraints of antibiotic production under natural environmental conditions. Environmental and other factors that suppress the antimicrobial action of antibiotics have to be studied to exploit the potential of antibiotics of PGPR in crop protection.

Effect of timing of application and population dynamics on the degree of biological control of Sclerotinia sclerotiorum by bacterial antagonists

Antagonistic Pseudomonas spp. (DF-41 and PA-23) were evaluated for inhibition of germination of ascospores, and for the effect of timing of application and its effect on biological control of Sclerotinia sclerotiorum (Lib.) de Bary, causal agent of stem rot of canola. Population dynamics were also assessed. In all studies, a petal inoculation technique was used. Significant inhibition (P < 0.05) of germination of ascospores was observed at both log 4 and log 8 cfu (colony forming units) ml(-1) of bacterial populations. In the population study, the pathogen had no significant effect (P < 0.05) on bacterial populations; however, a significant (P < 0.05) increase in bacterial populations was observed after 24 h and a decrease occurred between 96 and 120 h. Significant differences in disease severity (P < 0.05) were found with respect to timing of ascospore applications in the control treatments (ascospores only). One isolate completely suppressed disease when co-applied with ascospores, while only minor suppression occurred when applied 24 or 48 h after. Results from all studies indicate PA-23 and DF-41 to be effective biocontrol agents against S. sclerotiorum of canola and to have practical implications for biological control of this disease by bacteria in the field.

The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23

The aim of the current study was to determine how quorum sensing (QS) affects the production of secondary metabolites in Pseudomonas chlororaphis strain PA23. A phzR mutant (PA23phzR) and an N-acylhomoserine lactone (AHL)-deficient strain (PA23-6863) were generated that no longer inhibited the fungal pathogen Sclerotinia sclerotiorum in vitro. Both strains exhibited reduced pyrrolnitrin (PRN), phenazine (PHZ) and protease production. Moreover, phzA-lacZ and prnA-lacZ transcription was significantly reduced in PA23phzR and PA23-6863. As the majority of secondary metabolites are produced at the onset of stationary phase, we investigated whether cross-regulation occurs between QS and RpoS. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. Finally, we discovered that QS and RpoS have opposing effects on PA23 biofilm formation. While both QS-deficient strains produced little biofilm, the rpoS mutant showed enhanced biofilm production compared with PA23. Collectively, our findings indicate that QS controls diverse aspects of PA23 physiology, including secondary metabolism, RpoS and biofilm formation. As such, QS is expected to play a crucial role in PA23 biocontrol and persistence in the environment.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function.

Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode

Antifungal antibiotic from Pseudomonas chlororaphis isolate PA23 was identified as Phenazine using TLC and HPLC. Phenazine recorded the highest inhibition zone of 21 mm with 35.55% percent inhibition of mycelial growth of Pythium aphanidermatum over control. It had a significant effect on the hyphal morphology of P. aphanidermatum and on spore germination of Botryodiplodia theobromae and Alternaria solani. Disorganization of hyphal morphology of P. aphanidermatum includes vacuolization, cell content degeneration and hyphal lysis. Similarly interaction of phenazine with Rhizoctonia solani resulted in abnormal swelling of hyphal tips was noticed in the hyphal tips. Similarly the germination of sclerotia of Macrophomina phaseolina, R. solani and Sclerotium rolfsii were completely inhibited by phenazine at a concentration 50 μl. Incubation of the eggs of the root knot nematode Meloidogyne incognita in 30 μl concentration of phenazine, completely suppressed the hatching of juveniles.

Genetic Diversity and Phylogeny of Antagonistic Bacteria against Phytophthora nicotianae Isolated from Tobacco Rhizosphere

The genetic diversity of antagonistic bacteria from the tobacco rhizosphere was examined by BOXAIR-PCR, 16S-RFLP, 16S rRNA sequence homology and phylogenetic analysis methods. These studies revealed that 4.01% of the 6652 tested had some inhibitory activity against Phytophthora nicotianae. BOXAIR-PCR analysis revealed 35 distinct amplimers aligning at a 91% similarity level, reflecting a high degree of genotypic diversity among the antagonistic bacteria. A total of 25 16S-RFLP patterns were identified representing over 33 species from 17 different genera. Our results also found a significant amount of bacterial diversity among the antagonistic bacteria compared to other published reports. For the first time; Delftia tsuruhatensis, Stenotrophomonas maltophilia, Advenella incenata, Bacillus altitudinis, Kocuria palustris, Bacillus licheniformis, Agrobacterium tumefaciens and Myroides odoratimimus are reported to display antagonistic activity towards Phytophthora nicotianae. Furthermore, the majority (75%) of the isolates assayed for antagonistic activity were Gram-positives compared to only 25% that were Gram-negative bacteria.

Evaluation of different fungicides for control of fusarium head blight in wheat inoculated with 3ADON and 15ADON chemotypes of Fusarium graminearum in Canada

Abstract Fusarium head blight (FHB) continues to threaten the economic sustainability of many small grain producers in Manitoba by causing losses in grain yield and quality. In this 2-year study, four fungicides were tested on two spring wheat cultivars with different levels of resistance to FHB inoculated with 3ADON and 15ADON chemotypes of Fusarium graminearum. All fungicides (prothioconazole, tebuconazole, metconazole and prothioconazole+ tebuconazole) reduced FHB index, per cent Fusarium-damaged kernels (% FDK) and deoxynivalenol (DON) levels and increased yield compared with the non-sprayed control in the moderately resistant cultivar ‘Glenn’. In the highly susceptible cultivar ‘Roblin’, all fungicides reduced FHB index and increased yield compared with the non-sprayed control; however, inconsistent results were observed for FDK and DON. Differences were observed between the two chemotypes for all variables in both years except for FDK and yield in 2010. This study confirmed that host resistance plays an important role in host–pathogen–fungicide interaction. Therefore, the combined effect of growing moderately resistant cultivars with fungicide application can reduce damage caused by FHB even under high FHB severity.