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.

Identification of Antifungal Compounds from Avocado Rhizobacteria (Bacillus spp.) against Fusarium spp., by a Bioassay-Guided Fractionation Approach.

Antimicrobial compounds produced by bacteria have been increasingly acknowledged as an important resource for the control of phytopathogens. We used a bioassay-guided fractionation approach to identify antifungal metabolites produced by two avocado rhizobacteria (INECOL-4742 and INECOL-5927), both members of the Bacillus subtilis/B. amyloliquefaciens species complex, against Fusarium solani and F. kuroshium, causal agent of Fusarium dieback in avocado and other hosts. The butanol (BuOH) organic extract from INECOL-4742 (B1-Bu) exhibited the highest percentage of inhibition (PI) against F. solani (78.76 %), also inhibiting F. kuroshium by up to 44.30 %. Primary fractions, Bu-F3, Bu-F12 and Bu-F15, obtained by silica gel open column chromatography, exhibited the highest PI against F. solani (28.57 % to 33.50 %) and F. kuroshium (38.78 % to 45.00 %). The presence of cyclic lipopeptides from the iturin, surfactin and fengycin families in B1-Bu extracts and primary fractions was determined by UPLC-ESI-HRMS. The Confocal Laser Microscopy analysis revealed deformations in the hyphae of F. kuroshium exposed to extracts, primary fractions and C-13 surfactin chemical standard. These results emphasize the potential of natural products from Bacillus for the control of the emerging phytopathogenic fungus F. kuroshium.

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.

Phytophthora Root Rot Modifies the Composition of the Avocado Rhizosphere Microbiome and Increases the Abundance of Opportunistic Fungal Pathogens.

The structure and function of rhizosphere microbial communities are affected by the plant health status. In this study, we investigated the effect of root rot on the avocado rhizosphere microbiome, using 16S rDNA and ITS sequencing. Furthermore, we isolated potential fungal pathogens associated with root rot symptoms and assessed their pathogenic activity on avocado. We found that root rot did not affect species richness, diversity or community structure, but induced changes in the relative abundance of several microbial taxa. Root rot increased the proportion of Pseudomonadales and Burkholderiales in the rhizosphere but reduced that of Actinobacteria, Bacillus spp. and Rhizobiales. An increase in putative opportunistic fungal pathogens was also detected in the roots of symptomatic trees; the potential pathogenicity of Mortierella sp., Fusarium spp., Lasiodiplodia sp. and Scytalidium sp., is reported for the first time for the State of Veracruz, Mexico. Root rot also potentially modified the predicted functions carried out by rhizobacteria, reducing the proportion of categories linked with the lipid and amino-acid metabolisms whilst promoting those associated with quorum sensing, virulence, and antibiotic resistance. Altogether, our results could help identifying microbial taxa associated to the disease causal agents and direct the selection of plant growth-promoting bacteria for the development of biocontrol microbial consortia.

Antifungal potential of Lauraceae rhizobacteria from a tropical montane cloud forest against Fusarium spp.

The occurrence of pests and diseases can affect plant health and productivity in ecosystems that are already at risk, such as tropical montane cloud forests. The use of naturally occurring microorganisms is a promising alternative to mitigate forest tree fungal pathogens. The objectives of this study were to isolate rhizobacteria associated with five Lauraceae species from a Mexican tropical montane cloud forest and to evaluate their antifungal activity against Fusarium solani and F. oxysporum. Fifty-six rhizobacterial isolates were assessed for mycelial growth inhibition of Fusarium spp. through dual culture assays. Thirty-three isolates significantly reduced the growth of F. solani, while 21 isolates inhibited that of F. oxysporum. The nine bacterial isolates that inhibited fungal growth by more than 20% were identified through 16S rDNA gene sequence analysis; they belonged to the genera Streptomyces, Arthrobacter, Pseudomonas, and Staphylococcus. The volatile organic compounds (VOC) produced by these nine isolates were evaluated for antifungal activity. Six isolates (Streptomyces sp., Arthrobacter sp., Pseudomonas sp., and Staphylococcus spp.) successfully inhibited F. solani mycelial growth by up to 37% through VOC emission, while only the isolate INECOL-21 (Pseudomonas sp.) inhibited F. oxysporum. This work provides information on the microbiota of Mexican Lauraceae and is one of the few studies identifying forest tree-associated microbes with inhibitory activity against tree pathogens.

Avocado rhizobacteria emit volatile organic compounds with antifungal activity against Fusarium solani, Fusarium sp. associated with Kuroshio shot hole borer, and Colletotrichum gloeosporioides.

Recent studies showed that bacterial volatile organic compounds (VOCs) play an important role in the suppression of phytopathogens. The ability of VOCs produced by avocado (Persea americana Mill.) rhizobacteria to suppress the growth of common avocado pathogens was therefore investigated. We evaluated the antifungal activity of VOCs emitted by avocado rhizobacteria in a first screening against Fusarium solani, and in subsequent antagonism assays against Fusarium sp. associated with Kuroshio shot hole borer, Colletotrichum gloeosporioides and Phytophthora cinnamomi, responsible for Fusarium dieback, anthracnosis and Phytophthora root rot in avocado, respectively. We also analyzed the composition of the bacterial volatile profiles by solid phase microextraction (SPME) gas chromatography coupled to mass spectrometry (GC-MS). Seven isolates, belonging to the bacterial genera Bacillus and Pseudomonas, reduced the mycelial growth of F. solani with inhibition percentages higher than 20%. Isolate HA, related to Bacillus amyloliquefaciens, significantly reduced the mycelial growth of Fusarium sp. and C. gloeosporioides and the mycelium density of P. cinnamomi. Isolates SO and SJJ, also members of the genus Bacillus, reduced Fusarium sp. mycelial growth and induced morphological alterations of fungal hyphae whilst isolate HB, close to B. mycoides, inhibited C. gloeosporioides. The analysis of the volatile profiles revealed the presence of ketones, pyrazines and sulfur-containing compounds, previously reported with antifungal activity. Altogether, our results support the potential of avocado rhizobacteria to act as biocontrol agents of avocado fungal pathogens and emphasize the importance of Bacillus spp. for the control of emerging avocado diseases such as Fusarium dieback.

Ensayo de migración global en empaques para alimentos: evaluación de patrones internos alternativos / Overall migration test in food packaging: Evaluation of alternative internal standards / Ensaio de migração global em embalagens de alimentos: avaliação de padrões internos alternativos

Resumen En el ensayo de migración global para empaques plásticos destinados a alimentos grasos se emplea triheptadecanoato de glicerilo (GTM) como patrón interno para la cuantificación de los ésteres metílicos de ácidos grasos (FAMEs) por cromatografía de gases. En este trabajo se evaluaron 3-fenil propanoato de etilo (EFP) y ácido tetradecanóico (AM) como patrones internos alternativos. El uso de AM presentó comportamiento lineal (r > 0,99 y %Sb < 5%), buena precisión intermedia (HORRATr % 0,9) y exactitud (%R % 100,6%). En contraste, la evaluación del EFP, demostró que este no es un patrón interno adecuado para la cuantificación de FAMEs. De otra parte, se evaluó estadísticamente que no hay diferencias significativas en el cálculo de la migración global a partir de FAMEs cuantificados usando como patrón interno GTM o AM, mientras que el uso de EFP sí presenta diferencias significativas.

Abstract In the global migration test for plastic packaging intended for fatty foods, glyceryl triheptadecanoate (GTM) is used as the internal standard for the quantification of fatty acid methyl esters (FAMEs) by gas chromatography. In this work, ethyl 3-phenyl propanoate (EFP) and tetradecanoic acid (AM) were evaluated as alternative internal standards The use of AM presented linear behavior (r > 0.99 and %Sb < 5%), good intermediate precision (HORRATr % 0.9), and accuracy (%R % 100.6%). In contrast, the evaluation of EFP demonstrated that this is not an adequate internal standard for the quantification of FAMEs In addition, it was statistically evaluated that there are no significant differences in the calculation of the global migration from FAMEs quantified using either GTM or AM as internal standards, but the use of EFP presents significant differences.

Resumo No ensaio de migração global para embalagens plásticas destinadas aos alimentos gordurosos, é usado o triheptadecanoato de glicerina (GTM) como padrão interno para a quantificação dos ésteres metílicos de ácidos gordos (FAMEs) mediante cromatografia em fase gasosa. Neste papel foram avaliados 3-fenil propanoato de etilo (EFP) e ácido tetradecanóico (AM) como padrões internos alternativos O (AM) apresentou comportamento linear (r > 0,99 e %Sb < 5%), boa precisão intermédia (HORRATr % 0,9) e precisão (%R % 100,6%). Em contraste, a avaliação do (EFP), mostrou que este não é um padrão interno adequado para a quantificação de FAMEs Por outro lado, uma avaliação estatística indicou que não há diferenças significativas no cálculo da migração global de FAMEs quantificadas utilizando como padrão interno GTM ou AM, em contrapartida o uso do EFP apresenta diferenças significativas.

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.

Antifungal activity of avocado rhizobacteria against Fusarium euwallaceae and Graphium spp., associated with Euwallacea spp. nr. fornicatus, and Phytophthora cinnamomi.

Plant rhizobacteria have been successfully used as biocontrol agents against fungal phytopathogens. However, their potential to control two important avocado diseases, namely Fusarium dieback (FD) and Phytophthora root rot (PRR), has been poorly studied. FD is an emerging disease triggered by fungi associated with two ambrosia beetle species (Euwallacea fornicatus species complex), while PRR is caused by Phytophthora cinnamomi, a soil-borne oomycete. In the present work, the antifungal activity of bacteria isolated from avocado rhizosphere was tested in dual culture assays against Fusarium euwallaceae, Graphium euwallaceae and Graphium sp., causal agents of FD, and against P. cinnamomi. In 2015, rhizosphere soil samples of FD infested and non-infested avocado trees were collected from a commercial avocado orchard in Escondido, California. In an initial screening, 72 of the 168 assessed bacterial isolates reduced mycelial growth of F. euwallaceae by up to 46%. Eight bacterial isolates showing inhibition percentages larger than 40% were then selected for further antagonism assays against the other fungal pathogens. Five bacterial isolates, determined by 16S rDNA sequencing to belong to the Bacillus subtilis/Bacillus amyloliquefaciens species complex, successfully inhibited the mycelial growth of both Graphium species by up to 30%. The same isolates and an additional isolate identified as Bacillus mycoides, inhibited the growth of P. cinnamomi by up to 25%. This is the first report of avocado rhizobacteria with antifungal activity against pathogens responsible for FD and PRR in avocado.