Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing disease caused by the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the diffusible antibiotics phenazine-1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin (PRN). Because the individual contribution of these antibiotics to PA23 biocontrol has not been defined, mutants deficient in the production of phenazine (PHZ), PRN or both antibiotics were created. Analysis of the PHZ mutant revealed enhanced antifungal activity in vitro and wild-type levels of Sclerotinia disease suppression. Conversely, the PRN- and the PRN/PHZ-deficient strains exhibited decreased antifungal activity in vitro and markedly reduced the ability to control Sclerotinia infection of canola in the greenhouse. These findings suggest that PRN is the primary antibiotic mediating biocontrol of this pathogen. Analysis of prnA-lacZ and phzA-lacZ transcriptional fusions revealed that PRN and PHZ are not subject to autoregulation; moreover, they do not cross-regulate each other. However, HPLC showed a twofold increase in PRN levels in the PHZ(-) background. Finally, PHZ, but not PRN production, is involved in biofilm development in P. chlororaphis PA23.

Although many Bacillus species are known to be good antibiotic producers capable of acting as biocontrol agents, the underlying antimicrobial mechanisms are often poorly understood. In this study, 21 Bacillus strains, demonstrating over 50% mycelial inhibition against Sclerotinia sclerotiorum as well as significant control in plant assays, were examined for the presence of antibiotic biosynthetic genes. Primers specific for bacillomycin D, iturin A, surfactin, mycosubtilin, fengycin, and zwittermicin A were used to amplify biosynthetic genes from these bacteria using PCR. The majority of strains harbored surfactin (21/21) and iturin A (20/21) biosynthetic genes. Three strains (Bacillus subtilis 3057, Bacillus amyloliquefaciens BS6, and Bacillus mycoides 4079) were positive for bacillomycin D, whereas 4 strains (B. subtilis H-08-02, B. subtilis 3057, B. amyloliquefaciens BS6, and B. mycoides 4079) showed the presence of the fengycin biosynthetic gene. The zwittermicin A gene was detected in B. mycoides S, Bacillus thuringiensis BS8, and B. amyloliquefaciens BS6. Sequence analysis of purified PCR products revealed homology with corresponding genes from other Bacillus sp. in the GenBank database. Production of particular antibiotics in strains BS6, H-08-02, 3057, and 4079 was confirmed through matrix-assisted laser desorption ionization - time of flight - mass spectroscopy (MALDI-TOF-MS). This study revealed the equivalent capability of different Bacillus strains from various microhabitats to produce the above-mentioned antibiotics and highlights the possibility of using some strains as potential biocontrol agents under different microhabitats distant from their original habitat. Furthermore, it will enable researchers to develop rational strategies for the application of the antagonists and their metabolites within an agroecosystem. To the best of our knowledge, this is the first report of a B. mycoides strain that carries biosynthetic genes and produces fengycin and surfactin.

Cladonia is one of the largest lichen-forming ascomycete genera. It was formerly divided into ten sections, three of which, Crustaceae (Cladina), Tenues, and Impexae, are called the reindeer lichens. While previous studies have elucidated the relationships between species and sections, they often examined only one or a few specimens of each species in the analysis. This study examined the monophyly of selected members of sections Crustaceae, Tenues, and Impexae and their relationships in the genus Cladonia using the internal transcribed spacer region of the nuclear ribosomal DNA (ITS rDNA) and the mitochondrial small subunit gene of the mitochondrial ribosomal DNA (mtSSU). The phylogenetic tree contained four clades, two representing species in section Impexae, one representing species that belong to sections Crustaceae and Tenues, and one clade with C. arbuscula and related species. Five of 22 species, C. pycnoclada, C. stellaris, C. evansii, C. ciliata and C. subtenuis, showed monophyly in the phylogenetic tree; some of these 5 species have been shown previously to be monophyletic. The thallus branching pattern was interpreted as an important heritable character using the mtSSU network. Three duplets of paraphyletic species were further examined using ITS rDNA haplotype networks and AMOVA analysis. The results for the species duplets showed some mixing of haplotypes but the AMOVA analysis provided support for species separation within the duplets. While the evidence supports distinct species, further study is needed to conclusively show separate species in these duplets.

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine-and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P 0 0.05) root bacterial number and Sclerotinia wilt suppression (P 0 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P 0 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.

Recognition and defense responses are early events in plant-pathogen interactions and between lichen symbionts. The effect of elicitors on responses between lichen symbionts is not well understood. The objective of this study was to compare the difference in recognition- and defense-related gene expression as a result of culture extracts (containing secreted water-soluble elicitors) from compatible and incompatible interactions at each of 3 resynthesis stages in the symbionts of Cladonia rangiferina. This study investigated gene expression by quantitative PCR in cultures of C. rangiferina and its algal partner, Asterochloris glomerata/irregularis, after incubation with liquid extracts from cultures of compatible and incompatible interactions at 3 early resynthesis stages. Recognition-related genes were significantly upregulated only after physical contact, demonstrating symbiont recognition in later resynthesis stages than expected. One of 3 defense-related genes, chit, showed significant downregulation in early resynthesis stages and upregulation in the third resynthesis stage, demonstrating a need for the absence of chitinase early in thallus formation and a need for its presence in later stages as an algal defense reaction. This study revealed that recognition- and defense-related genes are triggered by components in culture extracts at 3 stages of resynthesis, and some defense-related genes may be induced throughout thallus growth. The parasitic nature of the interaction shows parallels between lichen symbionts and plant pathogenic systems.

Cladonia arbuscula and C. rangiferina are two reindeer lichen species, which are widely distributed in northern climates and have not been shown to be monophyletic. The wide species distribution may suggest an outcrossing reproductive life style, which may contribute to paraphyly and success of these two species. The current study examined the genetic diversity of mycobiont ascospore colonies within and between apothecia of two species, C. rangiferina and C. arbuscula s. l. using Randomly Amplified Polymorphic DNA (RAPD)-PCR. It also estimated gene flow, morphological variability, and colony growth rates. Cladonia rangiferina showed higher rates of growth than C. arbuscula but C. arbuscula showed more variability in growth between apothecia than C. rangiferina. Both species showed levels of genetic variation within and among apothecia that was consistent with heterothallism and recombination. AMOVA analyses provided evidence for gene flow among apothecia in both species. It further hypothesized that higher genetic variation in C. arbuscula than in C. rangiferina may give C. arbuscula an adaptive advantage over C. rangiferina.

It is widely recognized that the lichen is the product of a fungus and a photosynthetic partner (green alga or cyanobacterium) but its acceptance was slow to develop throughout history. The development of powerful microscopic and other lab techniques enabled better understanding of the interface between symbionts beginning with the contentious concept of the dual nature of the lichen thallus. Even with accelerating progress in understanding the interface between symbionts, much more work is needed to reach a level of knowledge consistent with that of other fungal interactions. This review describes the interface between algal and fungal symbionts in lichens, including the appearance of interacting symbionts, our current understanding of communication between symbionts, and emerging new concepts in light of the challenges and debates that have provided lichenology with an enriched history. Communication between symbionts occurs before physical contact, which has been studied by microscopy, movement of molecules between symbionts, and gene expression studies. New discoveries include the interaction with more than one alga in a lichen thallus as well as other unrelated fungi and bacteria typically found on or within the thallus. This mini review briefly describes our current state of knowledge and highlights new directions for further study.

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