The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

In the last 40 years, the concept of microorganisms that can enhance plant productivity matured into a research field, stretching from in vitro experiments, greenhouse and field trials done for research purposes, to commercial products applied worldwide. Currently numerous companies, big and small, are producing and marketing a large variety of seed inoculants and other microbial products for agricultural and horticultural uses (Bashan et al. 2014; Calvo et al. 2014). The market value of plant growth-promoting rhizobacteria/ bacteria (PGPR/PGPB), biological control agents, and a variety of biostimulants was valued at USD 6.00 Billion in 2016 and is projected to reach 14.65 Billion by 2023, growing at a compound annual growth rate of 13.80% from 2017 (https:// www.marketsandmarkets.com/Market-Reports/top-10-trendagricultural-biological-market-139215554.html). The scientific community engaging in research with these microorganisms is interested mainly in new developments, new products and new scientific concepts. As such, precise validation or simple confirmation of previous results or repetition of already published studies has very low priority. Because every scientific journal is interested in innovation, these results are seldom, or never, published. The direct user of such published information is the inoculant industry. Naturally because of intellectual property, issued information of failed experiments is mostly unavailable. Yet, quite a few studies repeated on commercial inoculants from various industries did not provide the benefits expected from these products (Denton et al. 2002; De Bruin et al. 2010; Karamanos et al. 2010). Such failures are better known in the biomedical industry (Petsko 2010). We identified four potential pitfalls responsible for such failures in the PGPR/PGPB research field; (i) improper description of fermentation processes by the original publication; (ii) improper description of consortia when used, (iii) improper description how consortia are made of, (iv) most times, the publications also don’t clearly disclose formulation techniques, exact dosage of microbial products used per unit area, soil physico-chemicals conditions etc., which could seriously impact the performance of microbial products, and (v) very few formal publications describing failure of a set of experiments (Bacilio et al. 2017; Karamanos et al. 2010; Martínez-Viveros et al. 2010). All PGPR/PGPB inoculants are culturable bacteria. Consequently, proper fermentation to produce large population of bacteria to be later formulated into an inoculant is one of the most essential features of inoculant production (Bashan et al. 2014; Calvo et al. 2014). Most publications followed the first original one on any PGPR/PGPB plant interaction and sometimes even in the first publication, to save printing space in the journal, the fermentation process is described briefly or as a reference from another PGPR/PGPB system following a statement “with small modifications” where the changes are described, but rarely a fully detailed protocol is provided. This first paper is commonly used as a reference for future publications on specific PGPR/PGPB interaction. While it is common that the experimental laboratory uses analytical ingredients for small fermentation studies, to save costs, the industrial manufacturers of inoculants use less purified ingredients. From numerous microbiological studies it is * Luz E. de-Bashan luz@bashanfoundation.org

Plant growth-promoting bacteria, such as Azospirillum brasilense, have the potential to significantly increase algal growth rates through a variety of mechanisms including the production of indole-3-acetic acid, an auxin hormone. A. brasilense promotion of growth in Chlorella sorokiniana is well-established for co-cultures suspended in alginate bead structures, however, its impacts on other types of green algae grown in suspended cultures is not well-understood. The objective of this research was to determine the impact of A. brasilense and indole-3-acetic acid on growth promotion and energy storage product accumulation in suspended cultures of C. sorokiniana and Auxenochlorella protothecoides. Suspended cultures were grown in lab-scale photobioreactors under the following conditions: algae grown on chemical medium (control), in co-culture with A. brasilense, chemical medium supplemented with exogenous indole-3-acetic acid, and algae grown on spent medium from A. brasilense. The results showed that co-cultures and exogenous indole-3-acetic acid stimulated growth in both algae types but the effect was stronger in C. sorokiniana. These same treatments also suppressed neutral lipids (particularly triacylglycerol) and starch during exponential growth of C. sorokiniana. Indole-3-acetic acid and co-cultures suppressed starch in A. protothecoides. Spent medium resulted in slight growth promotion in C. sorokiniana but significant growth suppression in A. protothecoides. It also led to significantly different compositional changes compared to using live A. brasilense, indicating that bioactive constituents in A. brasilense secretions are transient or that physical cell attachment is important for ensuring adequate mass transfer of these constituents. Overall, the findings suggest that indole-3-acetic acid and live A. brasilense mobilize cellular energy resources for growth.

Drought is one of the major worldwide causes of losses in agriculture and livestock farming, including the dry Caribbean zone in Colombia. Therefore, the objective was to determine if a synthetic community of droughttolerant plant growth-promoting bacteria (PGPB) isolated from the rhizosphere of Guinea grass (Megathyrsus maximus) could improve the plant's drought tolerance. The PGPB were divided into three groups (A, B, and C) according to their drought tolerance and used for the inoculation of Guinea grass seeds. The treatments consisted of seven combinations of different bacterial groups with non-inoculated watered and drought controls. Fortyday-old plants were exposed to 10 days without water, after which the plant biomass, two antioxidant enzymatic activities, proline accumulation, and membrane stability were determined. The results showed that drought stress significantly decreased the total biomass and membrane stability and increased the glutathione reductase (GR) activity and proline accumulation in non-inoculated plants. Group C bacterial strains were found to be more effective in alleviating drought stress than the other PGPB groups and were identified as Bacillus spp. Group C was evaluated in a 48-h drought-stress simulation using D-sorbitol on 20-day-old plantlets in which the antioxidant responses were measured. The D-sorbitol significantly increased the GR activity and proline accumulation in the non-inoculated plants. The plantlets that were inoculated with the Bacillus spp. group showed significantly reduced GR activity and increased proline accumulation. The results of the study suggested that GR activity and proline accumulation were key mechanisms used by Guinea grass to cope with drought stress. The results also showed that the co-inoculation of Bacillus spp. has the potential to ameliorate drought stress through the modulation of these mechanisms.

The effects of riboflavin and its degradation compound, lumichrome, produced by Azospirillum brasilense on the growth and production of carbohydrates, lipids, and pigments in Chlorella sorokiniana were analyzed. A. brasilense is able to produce and release riboflavin from the first 24 h of incubation in Synthetic Growth Medium (SGM). Synchronic peaks of lumichrome and riboflavin suggest that the former originates from the photodegradation of the latter. The constant growth of bacterial population indicated that riboflavin and lumichrome are cell density-independent. In this study, riboflavin showed a higher effect on the population growth of Chlorella than did lumichrome. The effects of synthetic riboflavin and lumichrome on microalgae were dependent on the concentration of these compounds in the culture media. Lower concentrations of riboflavin (1 ng mL(-1) or 10 ng mL(-1)) enhanced the growth of C. sorokiniana, while the highest amount of riboflavin (100 ng mL(-1)) showed no effect. Synthetic riboflavin and lumichrome significantly increased the production of carbohydrates compared to the basal medium. Lumichrome showed the highest production of carbohydrates at 48 h while riboflavin at 96 h. Pigments were significantly more affected by the presence of lumichrome than by riboflavin. Enhanced growth and production of carbohydrates and pigments in C. sorokiniana grown in exudates of A. brasilense, suggests a synergistic effect of the riboflavin and lumichrome produced and exuded by this bacterium.

Massive clear-cutting of wild stands of mesquite trees in the Mexican part of the Sonoran Desert result from high demand for this wood by the charcoal industry. Consequently, there is a need to develop techniques for reforestation of this tree in the desert and maintain its natural diversity at the same time. An outdoor nursery procedure to produce mesquite transplants from diversely originated seeds for reforestation of arid zones was developed. This procedure involved: 1) inoculation of the seedlings in the nursery with the plant growth-promoting bacteria (PGPB) Azospirillum brasilense immobilized in dry microbeads of alginate, and 2) developing a reliable way to monitor plant development and aerial volume in the nursery for the entire growth period of seven months before transplantation. Dry microbeads containing the PGPB and maintained at room temperature were tested for survival of bacteria for up to seven months. These dry microbeads maintained sufficient population levels of A. brasilense to inoculate the plant for the entire period. Inoculation with the PGPB enhanced all growth parameters of the plants, including biomass, aerial volume, root system, and chlorophyll pigments, but not the auxiliary photosynthetic pigments. The PGPB was specifically identified colonizing the roots of the transplants by fluorescent in situ hybridization for the entire growth period. Measuring a few simple parameters allowed development of a workable model for plant growth. This model was confirmed by data obtained from sacrificed plants whose parameters were measured directly. This study shows that outdoor nursery cultivation of inoculated mesquite transplants is feasible.

Resource islands (RIs), a natural revegetation phenomenon in arid lands, consist of a single nurse tree or few large shrubs and numerous understory nurslings. We analyzed 18 individual mesquite RIs for plant diversity and richness, area, trunk diameter (reflecting age), soil characteristics, physiological functionality of microbial populations, and interactions among these variables. Nursing Capacity reflected the availability of habitat and was positively correlated to plant richness, but not to plant diversity. No relationship between plant diversity and bacterial diversity was found. The structure of the bacterial communities of RIs differed from the bacterial communities of bare areas, which showed greater richness and diversity compared with those of RIs. The Nursing Capacity of the RIs was related to plant richness and accompanied by variations in soil properties. A high correlation was found by substrate utilization analysis between metabolic parameters of bacteria and diversity and richness of plants in the RIs. RI bacterial communities were more metabolically active and could degrade different carbon sources than bare area communities. RI bacterial communities contained species with greater capability to metabolize diverse carbon substrates in soil with more organic matter. Bacteria from low, medium, and high plant diversity areas were cultured and found to belong to four bacterial families. This study demonstrates that numerous parameters interact, but not every parameter significantly affected bacterial activity in the RI.

The adaptation and performance of orchid mycorrhizae in heavy metal-polluted soils have been poorly explored. In the present study, proteomic and metabolic approaches were used to detect physiological changes in orchid roots established in a heavy metal-polluted soil and to ascertain whether mycorrhizal fungi affect the metabolic responses of roots. Young Bipinnula fimbriata plantlets were established in control and heavy metal-polluted soils in a greenhouse. After 14 months, exudation of root organic acids, phenolics, percentage of mycorrhization, mineral content, and differential protein accumulation were measured. More root biomass, higher root colonization, and higher exudation rates of citrate, succinate, and malate were detected in roots growing in heavy metal-polluted soils. Higher accumulation of phosphorus and heavy metals was found inside mycorrhizal roots under metal stress. Under non-contaminated conditions, non-mycorrhizal root segments showed enhanced accumulation of proteins related to carbon metabolism and stress, whereas mycorrhizal root segments stimulated protein synthesis related to pathogen control, cytoskeleton modification, and sucrose metabolism. Under heavy metal stress, the proteome profile of non-mycorrhizal root segments indicates a lower induction of defense mechanisms, which, together with the stimulation of enzymes related to carotenoid biosynthesis and cell wall organization, may positively influence mycorrhizal fungi colonization. The results point to different metabolic strategies in mycorrhizal and non-mycorrhizal root segments that are exposed to heavy metal stress. The results indicate that root colonization by mycorrhizal fungi is stimulated to alleviate the negative effects of heavy metals in the orchids.

Soil degradation is an ecological disturbance, usually human‐caused, that negatively affects the vegetation and climate of an ecosystem, particularly arid and semiarid environments. These degraded soils can be restored by using native perennial plants inoculated with specific microorganisms. We studied the changes in root growth and the rhizosphere bacterial community of mesquite seedlings (Prosopis articulata) after inoculation with the endophytic bacteria Bacillus pumilus ES4, over 3 cycles of growth in the same soil under desert climatic conditions, and found that inoculation significantly enhanced root biomass during the growth cycles but not shoot biomass or root and shoot lengths. Fluorescent in situ hybridization analysis demonstrated that B. pumilus colonized the root cap, apical meristem, and elongation zone, forming small colonies, on roots from soil‐grown mesquite. Inoculation also significantly changed the bacterial community structure of rhizophere and nonrhizosphere (without plants) soils based on denaturing gradient gel electrophoresis profiles. The changes were highly stable, and the bacterial community structure was maintained throughout the experimental period and not affected by plant replacement. The 16S rRNA pyrosequencing confirmed the changes on structure of bacterial community and revealed an impact on the top taxonomic levels analyzed. The rhizospheres of inoculated plants showed a significant increase in the abundance of Proteobacteria and Acidobacteria coupled with a concomitant decrease in Actinobacteria, whereas an opposite response was observed in nonrhizospheric degraded soils. Overall, inoculation with B. pumilus reduced bacterial diversity but increased the Rhizobium population in the soil. The class Bacilli, despite B. pumilus inoculum, showed minimal variation.

The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth–promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

The capacity of pepper plants to alleviate salt stress when inoculated with the plant growth-promoting bacteria (PGPB) Pseudomonas stuzeri and/or supplementation with humic acids was compared under in vitro conditions in a growth chamber (three independent experiments), greenhouse (four experiments) and field conditions (two experiments). Although inoculation with PGPB or humic acids significantly mitigated negative effects of salinity on germinating pepper seedlings under in vitro conditions, the effect was far less marked under greenhouse conditions and almost non-existent under field conditions, having no impact on yield of peppers under saline conditions. This study demonstrates that the improvement in growth with PGPB and humic acids in vitro may not be scalable from the laboratory to greenhouse and to field conditions. Not all PGPB improvements scale from the laboratory, greenhouse to field conditions and that the potential that laboratory results may not scale is a factor to be considered in this research field.

Little is known about Orchidaceae plants in Chile and their mycorrhizal associations, a key issue for designing protective actions for endangered species. We investigated root fungi from seven terrestrial orchid species to identify potential mycorrhizal fungi. The main characteristics of Rhizoctonia-like fungi were observed under light microscopy, and isolates were identified through PCR-ITS sequencing. Molecular identification of fungal sequences showed a high diversity of fungi colonizing roots. Fungal ability to germinate seeds of different orchids was determined in symbiotic germination tests; 24 fungal groups were isolated, belonging to the genera Tulasnella, Ceratobasidium, and Thanatephorus. Furthermore, dark septate and other endophytic fungi were identified. The high number of Rhizoctonia-like fungi obtained from adult orchids from the Coastal mountain range suggests that, after germination, these orchids may complement their nutritional demands through mycoheterotrophy. Nonetheless, beneficial associations with other endophytic fungi may also co-exist. In this study, isolated mycorrhizal fungi had the ability to induce seed germination at different efficiencies and with low specificity. Germin ation rates were low, but protocorms continued to develop for 60 days. A Tulasnella sp. isolated from Chloraea gavilu was most effective to induce seed germination of different species. The dark septate endophytic (DSE) fungi did not show any effect on seed development; however, their widespread occurrence in some orchids suggests a putative role in plant establishment.

Eight greenhouse experiments were performed to compare the effect of seven plant growth-promoting (rhizo)bacteria (PGPR/PGPB) on fresh and dry weights of four crop plants. This has been done to validate if fresh weight measurements of plant variables can serve as reliable values when reporting the effect of these bacteria on plant growth. These experiments show that the growth promotion effects by the tested PGPR/PGPB, including Bacillus amyloliquefaciens GB03, Bacillus subtilis IN-937B, Bacillus altitudinis INR7, and Pseudomonas mandelii 89B-27 in corn and cucumber and Azospirillum brasilense Cd, A. brasilense Sp 245, and Azospirillum lipoferum Br 17 in pepper and tomato, varied significantly between fresh and dry weights of shoot, root, and/or whole plant in the repeated greenhouse experiments. These results support our hypothesis that using fresh weight determination for assessing plant growth promotion by beneficial bacteria is inherently faulty. Therefore, it is recommended that dry weight determination rather than fresh weight determination is used for plant growth promotion tests.

Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.

Restoration of degraded desert soil with three species of legume trees and the giant cardon cactus was evaluated 11 years after planting in the southern Sonora Desert.

Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR).

Strains of Bacillus subtilis are plant growth-promoting bacteria (PGPB) of many crops and are used as inoculants. PGPB colonization is an important trait for success of a PGPB on plants. A specific probe, based on the 16 s rRNA of Bacillus subtilis, was designed and evaluated to distinguishing, by fluorescence in situ hybridization (FISH), between this species and the closely related Bacillus amyloliquefaciens. The selected target for the probe was between nucleotides 465 and 483 of the gene, where three different nucleotides can be identified. The designed probe successfully hybridized with several strains of Bacillus subtilis, but failed to hybridize not only with B. amyloliquefaciens, but also with other strains such as Bacillus altitudinis, Bacillus cereus, Bacillus gibsonii, Bacillus megaterium, Bacillus pumilus; and with the external phylogenetic strains Azospirillum brasilense Cd, Micrococcus sp. and Paenibacillus sp. The results showed the specificity of this molecular probe for B. subtilis.

This study measured the relations between tryptophan production, the phytohormone indole-3-acetic acid (IAA) and the metabolism and accumulation of starch during synthetic mutualism between the microalgae Chlorella sorokiniana and the microalgae growth-promoting bacteria Azospirillum brasilense, created by co-immobilization in alginate beads. Experiments used two wild-type A. brasilense strains (Cd and Sp6) and an IAA-attenuated mutant (SpM7918) grown under nitrogen-replete and nitrogen-starved conditions tested under dark, heterotrophic and aerobic growth conditions. Under all incubating conditions, C. sorokiniana, but not A. brasilense, produced tryptophan. A significant correlation between IAA-production by A. brasilense and starch accumulation in C. sorokiniana was found, since the IAA-attenuated mutant was not producing increased starch levels. The highest ADP-glucose pyrophosphorylase (AGPase) activity, starch content and glucose uptake were found during the interaction of A. brasilense wild type strains with the microalgae. When the microalgae were grown alone, they produced only small amounts of starch. Supplementation with synthetic IAA to C. sorokiniana grown alone enhanced the above parameters, but only transiently. Activity of α-amylase decreased under nitrogen-replete conditions, but increased under nitrogen-starved conditions. In summary, this study demonstrated that, during synthetic mutualism, the exchange of tryptophan and IAA between the partners is a mechanism that governs several changes in starch metabolism of C. sorokiniana, yielding an increase in starch content.