Biocontrol and plant growth promoting traits of two avocado rhizobacteria are orchestrated by the emission of diffusible and volatile compounds.

Avocado (Persea americana Mill.) is a tree crop of great social and economic importance. However, the crop productivity is hindered by fast-spreading diseases, which calls for the search of new biocontrol alternatives to mitigate the impact of avocado phytopathogens. Our objectives were to evaluate the antimicrobial activity of diffusible and volatile organic compounds (VOCs) produced by two avocado rhizobacteria (Bacillus A8a and HA) against phytopathogens Fusarium solani, Fusarium kuroshium, and Phytophthora cinnamomi, and assess their plant growth promoting effect in Arabidopsis thaliana. We found that, in vitro, VOCs emitted by both bacterial strains inhibited mycelial growth of the tested pathogens by at least 20%. Identification of bacterial VOCs by gas chromatography coupled to mass spectrometry (GC-MS) showed a predominance of ketones, alcohols and nitrogenous compounds, previously reported for their antimicrobial activity. Bacterial organic extracts obtained with ethyl acetate significantly reduced mycelial growth of F. solani, F. kuroshium, and P. cinnamomi, the highest inhibition being displayed by those from strain A8a (32, 77, and 100% inhibition, respectively). Tentative identifications carried out by liquid chromatography coupled to accurate mass spectrometry of diffusible metabolites in the bacterial extracts, evidenced the presence of some polyketides such as macrolactins and difficidin, hybrid peptides including bacillaene, and non-ribosomal peptides such as bacilysin, which have also been described in Bacillus spp. for antimicrobial activities. The plant growth regulator indole-3-acetic acid was also identified in the bacterial extracts. In vitro assays showed that VOCs from strain HA and diffusible compounds from strain A8a modified root development and increased fresh weight of A. thaliana. These compounds differentially activated several hormonal signaling pathways involved in development and defense responses in A. thaliana, such as auxin, jasmonic acid (JA) and salicylic acid (SA); genetic analyses suggested that developmental stimulation of the root system architecture by strain A8a was mediated by the auxin signaling pathway. Furthermore, both strains were able to enhance plant growth and decreased the symptoms of Fusarium wilt in A. thaliana when soil-inoculated. Collectively, our results evidence the potential of these two rhizobacterial strains and their metabolites as biocontrol agents of avocado pathogens and as biofertilizers.

Electroantennographic Responses of Wild and Laboratory-Reared Females of Xyleborus affinis Eichhoff and Xyleborus ferrugineus (Fabricius) (Coleoptera: Curculionidae: Scolytinae) to Ethanol and Bark Volatiles of Three Host-Plant Species.

Chemical ecology studies on ambrosia beetles are typically conducted with either wild or laboratory-reared specimens. Unlike laboratory-reared insects, important aspects that potentially influence behavioral responses, such as age, physiological state, and prior experience are unknown in wild specimens. In this study, we compared the electroantennographic (EAG) responses of laboratory-reared and wild X. affinis and X. ferrugineus to 70% ethanol and bark odors (host kairomones) of Bursera simaruba, Mangifera indica, and Persea schiedeana aged for 2, 24, and 48 h. Chemical analyses of each odor treatment (bark species x length of aging) were performed to determine their volatilome composition. EAG responses were different between laboratory-reared and wild X. ferrugineus when exposed to ethanol, whereas wild X. affinis exhibited similar EAG responses to the laboratory-reared insects. Ethanol elicited the strongest olfactory responses in both species. Among the bark-odors, the highest responses were triggered by B. simaruba at 48 h in X. affinis, and P. schiedeana at 24 and 48 h in X. ferrugineus. Volatile profiles varied among aged bark samples; 3-carene and limonene were predominant in B. simaruba, whereas α-copaene and α-cubebene were abundant in P. schiedeana. Further studies are needed to determine the biological function of B. simaruba and P. schiedeana terpenes on X. affinis and X. ferrugineus, and their potential application for the development of effective lures.

Plant growth-promoting and non-promoting rhizobacteria from avocado trees differentially emit volatiles that influence growth of Arabidopsis thaliana.

Microbial volatile organic compounds (mVOCs) play important roles in inter- and intra-kingdom interactions, and they are also important as signal molecules in physiological processes acting either as plant growth-promoting or negatively modulating plant development. We investigated the effects of mVOCs emitted by PGPR vs non-PGPR from avocado trees (Persea americana) on growth of Arabidopsis thaliana seedlings. Chemical diversity of mVOCs was determined by SPME-GC-MS; selected compounds were screened in dose-response experiments in A. thaliana transgenic lines. We found that plant growth parameters were affected depending on inoculum concentration. Twenty-six compounds were identified in PGPR and non-PGPR with eight of them not previously reported. The VOCs signatures were differential between those groups. 4-methyl-2-pentanone, 1-nonanol, 2-phenyl-2-propanol and ethyl isovalerate modified primary root architecture influencing the expression of auxin- and JA-responsive genes, and cell division. Lateral root formation was regulated by 4-methyl-2-pentanone, 3-methyl-1-butanol, 1-nonanol and ethyl isovalerate suggesting a participation via JA signalling. Our study revealed the differential emission of volatiles by PGPR vs non-PGPR from avocado trees and provides a general view about the mechanisms by which those volatiles influence plant growth and development. Rhizobacteria strains and mVOCs here reported are promising for improvement the growth and productivity of avocado crop.

Diffusible and volatile organic compounds produced by avocado rhizobacteria exhibit antifungal effects against Fusarium kuroshium.

Rhizobacteria emit bioactive metabolites with antifungal properties that could be used for biocontrol of fungal diseases. In this study, we evaluated the potential of diffusible and volatile organic compounds (VOCs) emitted by avocado rhizobacteria to inhibit the growth of Fusarium kuroshium, one of the causal agents of Fusarium dieback (FD) in avocado. Three bacterial isolates (INECOL-6004, INECOL-6005, and INECOL-6006), belonging to the Bacillus genus, were selected based on their capacity to inhibit several avocado fungal pathogens, and tested in antagonism assays against F. kuroshium. The three bacterial isolates significantly inhibited F. kuroshium mycelial growth by up to 48%. The composition of bacterial diffusible compounds was characterized by the analysis of EtOAc and n-BuOH extracts by using ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS). The three bacterial isolates produced cyclo-lipopeptides belonging to the iturin, fengycin, and surfactin families. The antifungal activity of n-BuOH extracts was larger than that of EtOAc extracts, probably due to the greater relative abundance of fengycin in the former than in the latter. In addition, isolates INECOL-6004 and INECOL-6006 significantly inhibited F. kuroshium mycelial growth through VOC emission by up to 69.88%. The analysis of their VOC profiles by solid phase micro-extraction (SPME) coupled to gas chromatography and mass spectrometry (GC-MS) revealed the presence of ketones and pyrazine compounds, particularly of 2-nonanone, which was not detected in the VOC profile of isolate INECOL-6005. These results emphasize the need to further investigate the antifungal activity of each bioactive compound for the development of new formulations against fungal phytopathogens.

Plant growth-promoting rhizobacteria associated with avocado display antagonistic activity against Phytophthora cinnamomi through volatile emissions.

Rhizobacteria associated with crops constitute an important source of potentially beneficial microorganisms with plant growth promoting activity or antagonistic effects against phytopathogens. In this study, we evaluated the plant growth promoting activity of 11 bacterial isolates that were obtained from the rhizosphere of healthy avocado trees and from that of avocado trees having survived root rot infestations. Seven bacterial isolates, belonging to the genera Bacillus, Pseudomonas and Arthrobacter, promoted in vitro growth of Arabidopsis thaliana. These isolates were then tested for antagonistic activity against Phytophthora cinnamomi, in direct dual culture assays. Two of those rhizobacterial isolates, obtained from symptomatic-declining trees, displayed antagonistic activity. Isolate A8a, which is closely related to Bacillus acidiceler, was also able to inhibit P. cinnamomi growth in vitro by 76% through the production of volatile compounds. Solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS) allowed to tentatively identify the main volatiles emitted by isolate A8a as 2,3,5-trimethylpyrazine, 6,10-dimethyl-5,9-undecadien-2-one and 3-amino-1,3-oxazolidin-2-one. These volatile compounds have been reported to show antifungal activity when produced by other bacterial isolates. These results confirm the significance of rhizobacteria and suggest that these bacteria could be used for biocontrol of soil borne oomycetes through their volatiles emissions.