Combined inoculation with dark septate endophytes and arbuscular mycorrhizal fungi: synergistic or competitive growth effects on maize?

BACKGROUND: Effects on maize were assessed of dual inoculation with arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) isolated from other plant species. METHODS: Suspensions of DSE isolated from Stipa krylovii were prepared at different densities (2, 4, and 8 × 105 CFU mL- 1) and inoculated separately (AMF or DSE) or together (AMF + DSE), to explore their effects on maize growth. RESULTS: Inoculation with AMF or medium and high densities of DSE and combined inoculation (AMF + DSE) increased plant above-ground growth and altered root morphology. Differences in plant growth were attributable to differences in DSE density, with negative DSE inoculation responsiveness at low density. AMF promoted plant above-ground growth more than DSE and the high density of DSE promoted root development more than AMF. Combined inoculation might lead to synergistic growth effects on maize at low density of DSE and competitive effects at medium and high DSE densities. CONCLUSIONS: AMF and DSE co-colonized maize roots and they had positive effects on the host plants depending on DSE density. These findings indicate the optimum maize growth-promoting combination of AMF and DSE density and provide a foundation for further exploration of potentially synergistic mechanisms between AMF and DSE in physiological and ecological effects on host plants.

Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria.

BACKGROUND: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production. RESULTS: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion. CONCLUSIONS: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.

Next-generation biofertilizers and novel biostimulants: documentation and validation of mechanism of endophytic plant growth-promoting rhizobacteria in tomato.

A study was conducted to determine the suitability of the endophytes as probable next-generation biofertilizers and novel biostimulants. Enterobacter turicensis RCT5 and Stenotrophomonas maltophilia RCT31 showed a zone of solubilization, of phosphate, potassium, silicate, and zinc, produced phytase. Among the three media used for phosphate solubilization, the rhizospheric medium turned out to be the best-producing results in less than 24 h, while others took a longer time to give the same results. The strains exhibited differential ability to produce organic acids in the plate assay and eight of these were profuse producers of exopolysaccharides. We were able to partially elucidate the mechanism of solubilization of insoluble salts that included organic acids and protein activity in the cell-free culture filtrates of endophytes. All the root nodule endophytes showed potential as novel biostimulants and next-generation biofertilizers as found in the germination assay of tomato, a non-host crop using different methodologies. It proved that the endophytes have different mechanism of expressions of their plant growth-promoting traits as well as can promote the growth of tomato plant irrespective of the method used.

Effects of Tall Fescue Endophyte Type and Dopamine Receptor D2 Genotype on Cow-Calf Performance during Late Gestation and Early Lactation.

Grazing endophyte-infected, toxic tall fescue reduces cow/calf production; therefore, this study examines alternate strategies such as use of novel endophyte fescue varieties during late gestation and early lactation or genetic selection of resistant cows. Pregnant cows (n = 75) were randomly assigned to fescue endophyte type: 1) endophyte-infected ergot alkaloid producing tall fescue (E+) or 2) novel endophyte-infected, non-toxic tall fescue (NOV) within maternal (A|A, n = 38 and G|G, n = 37) DRD2 genotype to examine changes in cow/calf performance and milk production during late gestation and early lactation. Grazing E+ fescue pastures during late gestation reduced cow body weight gain but did not alter calf birth weight compared to NOV. Milk production and calf ADG during the first 30 day of lactation were lower for E+ than NOV. The calving rate was reduced, but not calving interval for E+ cows. The adjusted 205-day weight of calves was lower in those grazing E+ with their dams compared to NOV. There were no interactions between DRD2 genotype and fescue endophyte type indicating that genotype was not associated with response to E+ fescue in this study. Overall, grazing NOV tall fescue pastures rather than E+ during critical stages of production improved cow gain during late gestation, calving rate, early milk production and calf growth.

Bacterial endophytes: Molecular interactions with their hosts.

Plant growth promotion has been found associated with plants on the surface (epiphytic), inside (endophytic), or close to the plant roots (rhizospheric). Endophytic bacteria mainly have been researched for their beneficial activities in terms of nutrient availability, plant growth hormones, and control of soil-borne and systemic pathogens. Molecular communications leading to these interactions between plants and endophytic bacteria are now being unrevealed using multidisciplinary approaches with advanced techniques such as metagenomics, metaproteomics, metatranscriptomics, metaproteogenomic, microRNAs, microarray, chips as well as the comparison of complete genome sequences. More than 400 genes in both the genomes of host plant and bacterial endophyte are up- or downregulated for the establishment of endophytism and plant growth-promoting activity. The involvement of more than 20 genes for endophytism, about 50 genes for direct plant growth promotion, about 25 genes for biocontrol activity, and about 10 genes for mitigation of different stresses has been identified in various bacterial endophytes. This review summarizes the progress that has been made in recent years by these modern techniques and approaches.

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi.

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala-a triploid hybrid species-were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

Molecular insight into the endophytic growth of Beauveria bassiana within Phaseolus vulgaris in the presence or absence of Tetranychus urticae.

Entomopathogenic fungi are an important factor in the natural regulation of arthropod populations. Moreover, some can exist as an endophyte in many plant species and establish a mutualistic relationship. In this study, we have investigated the endophytic growth of Beauveria bassiana within different tissues of Phaseolus vulgaris in the presence and absence of Tetranuychus urticae. After the colonization of the B. bassiana within the internal tissues of P. vulgaris. The susceptibility of T. urticae appeared to depend on the life stage where high, moderate, and low mortalities were recorded among adults, nymphs, and eggs, respectively. In addition, this study provided, for the first time, molecular insight into the endophytic growth of B. bassiana by analyzing the expression of several genes involved in the development of the entomopathogenic fungi at 0-, 2-, and 7- days post-inoculation. B. bassiana displayed preferential tissue colonization within P. vulgaris that can be put into the following order based on the detection rate: leaf > stem > root. After analyzing the development-implicated genes (degrading enzymes, sugar transporter, hydrophobins, cell wall synthesis, secondary metabolites, stress management), the most remarkable finding is the detection of behavioral change between parasitic and endophytic Beauveria during post-penetration events. This study elucidates the tri-trophic interaction between fungus-plant-herbivore.

Bacterial and fungal endophyte communities in healthy and diseased oilseed rape and their potential for biocontrol of Sclerotinia and Phoma disease.

Phoma stem canker (caused by the ascomycetes Leptosphaeria maculans and Leptosphaeria biglobosa) is an important disease of oilseed rape. Its effect on endophyte communities in roots and shoots and the potential of endophytes to promote growth and control diseases of oilseed rape (OSR) was investigated. Phoma stem canker had a large effect especially on fungal but also on bacterial endophyte communities. Dominant bacterial genera were Pseudomonas, followed by Enterobacter, Serratia, Stenotrophomonas, Bacillus and Staphylococcus. Achromobacter, Pectobacter and Sphingobacterium were isolated only from diseased plants, though in very small numbers. The fungal genera Cladosporium, Botrytis and Torula were dominant in healthy plants whereas Alternaria, Fusarium and Basidiomycetes (Vishniacozyma, Holtermaniella, Bjerkandera/Thanatephorus) occurred exclusively in diseased plants. Remarkably, Leptosphaeria biglobosa could be isolated in large numbers from shoots of both healthy and diseased plants. Plant growth promoting properties (antioxidative activity, P-solubilisation, production of phytohormones and siderophores) were widespread in OSR endophytes. Although none of the tested bacterial endophytes (Achromobacter, Enterobacter, Pseudomonas, Serratia and Stenotrophomonas) promoted growth of oilseed rape under P-limiting conditions or controlled Phoma disease on oilseed rape cotyledons, they significantly reduced incidence of Sclerotinia disease. In the field, a combined inoculum consisting of Achromobacter piechaudii, two pseudomonads and Stenotrophomonas rhizophila tendencially increased OSR yield and reduced Phoma stem canker.

Isolation and characterization of endophytic bacteria for controlling root rot disease of Chinese jujube.

AIM: Fusarium oxysporum is the primary pathogen causing root rot disease that severely affects cultivation of jujube fruit in the Xinjiang province of China. The aim of this study was to identify endophytic bacteria in healthy jujube organs that could effectively suppress F. oxysporum growth. METHODS AND RESULTS: Different plant organs (leaves, twigs and roots) were collected from healthy Chinese jujube cultivated in southern Xinjiang province of China. The endophytic bacterium Brevibacterium halotolerans JZ7 was selected for its strong antagonistic activity and growth-promoting characteristics. Gas-chromatography mass-spectrometry analysis showed that acetoin, 2,3-butanediol and fenretinide were the three dominant volatile organic compounds produced by strain JZ7. Fenretinide strongly suppressed spore germination of F. oxysporum in vitro. Pot experiments showed that strain JZ7 colonized both the roots and rhizosphere soil of Chinese jujube and significantly reduced F. oxysporum level in jujube rhizosphere soil. CONCLUSION: We demonstrated that B. halotolerans JZ7 can be developed into a biological control agent to combat root rot disease of Chinese jujube in the Xinjiang province of China. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested strategy for biological control of jujube root rot disease is fully in accordance with the current principles of sustainability.

Studying Seed Microbiomes.

Recent studies indicate that seed microbiomes affect germination and plant performance. However, the interplay between seed microbiota and plant health is still poorly understood. To get a complete picture of the system, a comprehensive analysis is required, comprising culture-dependent and culture-independent techniques. In this chapter, we provide a combination of methods that are established and optimized for the analysis of the seed microbiome. These include methods to: (1) activate and cultivate dormant seed microbiota, (2) analyze microbiota in germinated seeds (with and without substrate), (3) quantify microbial DNA via real-time PCR, (4) deplete host DNA for amplicon and metagenome analysis, and (5) visualize seed endophytes in microtomed sections using fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). A deep understanding of the seed microbiome and its functions can help in developing new seed treatments and breeding strategies for sustainable agriculture.

Sampling of Plant Material to Study Endophytes in Small, Large, and Woody Plants.

The study of fungal, bacterial, and other endophytic microorganisms using high throughput DNA sequencing requires sampling and processing of plant material that eliminates phylloplane microorganisms and retains those inside the plant compartment. Leaves, stems, roots, and other plant tissues are removed from the plant, washed, surface sterilized, and stored for downstream applications. Especially in ecological studies, field work for sample collection may take place in remote locations where laboratory equipment and resources are rudimentary, and accessing samples from target plants can be challenging. This chapter serves as a guide to basic protocols in the design and sample collection for studies focused on the endophytes of leaf, stem, and root tissues.

Plant-associated Fungi: Methods for Taxonomy, Diversity, and Bioactive Secondary Metabolite Bioprospecting.

Plants harbor a large reservoir of fungal diversity, encompassing endophytic, epiphytic, phytopathogenic, and rhizosphere-associated fungi. Despite this diversity, relatively few fungal species have been characterized as sources of bioactive secondary metabolites. The role of secondary metabolites is still not fully understood; however, it is suggested that these metabolites play important roles in defense mechanisms and fungal interactions with other organisms. Hence, fungal secondary metabolites have potential biotechnological applications as prototype molecules for the development of therapeutic drugs. In this chapter, we describe the main methods used for routine fungi isolation, production of crude fungal extracts, and chemical characterization of bioactive compounds. In addition, explicative notes about the steps described are provided to explore the diversity of the endophytic, phytopathogenic, epiphytic, and rhizosphere fungi and to evaluate the biotechnological potential of each group.

Biocontrol of Tomato Bacterial Wilt by Foliar Spray Application of a Novel Strain of Endophytic Bacillus sp.

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4| |weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

Impact of endophyte inoculation on the morphological identity of cultivars of Lolium perenne (L) and Festuca arundinacea (Schreb.).

Grass endophytes have been shown to confer enhanced environmental resilience to symbiont cultivars with reports of modified growth. If inoculating with an endophyte (E+) made an accession morphologically distinct from its registered endophyte free (E-) accession, there could be protection and ownership issues for testing authorities and breeders. This study investigated if, in official Plant Breeders Rights (PBR) field trials, the morphological characteristics of E+and E- accessions of perennial ryegrass and tall fescue cultivars were sufficiently modified to designate them as mutually distinct and also distinct from their definitive accessions (Def), held by the testing authorities. Testing perennial ryegrass on 17 characters at 2 sites generated 48,960 observations and for tall fescue on 9 characters at 1 site, 12,960 observations (each for 3 accessions of 4 cultivars × 60 plants × 2 growing cycles). Distinctness required a p < 0.01 difference in a single character from the combined over years analysis (COYD). A few significant differences were recorded between E- and E+accessions. Cultivar Carn E+ was smaller than Carn E- for Infloresence Length (p < 0.01) in both years but COYD analysis (p < 0.05) was insufficient to declare distinctiveness. Overall, the number of observed differences between E-/E+ accessions was less or similar to the number expected purely by chance. In contrast, comparisons between Def and E- or E+ accessions showed a number of significant differences that were substantially more numerous than expected by chance. These results showed no conclusive evidence of endophyte inclusion creating false PBR distinctions but unexpectedly, several E- and E+ accessions were distinguished from their official definitive stock.

Isolation and characterization of Bacillus subtilis strain 1-L-29, an endophytic bacteria from Camellia oleifera with antimicrobial activity and efficient plant-root colonization.

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana.

[Promoting tanshinone synthesis of Salvia miltiorrhiza root by a seed endophytic fungus, Phoma herbarum D603].

The interaction of endophytes and host plant is an effective mean to regulate the growth and secondary metabolism of medicinal plants. Here we want to elucidate the effects and mechanism of Phoma herbarum D603 on the root development and tanshinone synthesis in root of Salvia miltiorrhiza by endophyte-plant coculture system. The mycelium of P. herbarum D603 was colonized in the root tissue space, and formed a stable symbiotic relationship with host plant. The in vitro activities analysis showed that the concentration of IAA produced by D603 can reach(6.45±0.23) µg·mL~(-1), and this strain had some abilities of phosphorus solubilization and siderophore production activities. The coculture experiment showed that strain D603 can significantly promote the synthesis and accumulation of tanshinones in the root of S. miltiorrhiza, in which after 8 weeks of treatment with D603, the content of tanshinone Ⅱ_A in the roots reached up to(1.42±0.59) mg·g~(-1). By the qRT-PCR analysis results, we found that D603 could improve the expression levels of some key genes(DXR, DXS, GGPP, HMGR, CPS) of tanshinone biosynthesis pathway in host plant S. miltiorrhiza, but the promoting effect mainly occurred in the early stage of the interaction, and the enzyme activity level decreased in varying degrees of the later stage. In summary, seed-associated endophyte P. herbarum D603 can promote the growth and root development of S. miltiorrhiza by producing hormones, promoting nutrient absorption and siderophore production, and promote the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway in S. miltiorrhiza.

Molecular crosstalk between the endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum - Modulation of lipoxygenase activity and beauvericin production during the interaction.

Plants comprise many asymptomatic fungal endophytes with potential roles of plant protection against abiotic and biotic stresses. Endophytes communicate with their host plant, with other endophytes and with invading pathogens but their language remains largely unknown. This work aims at understanding the chemical communication and physiological interactions between the fungal endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum. Oxylipins, common means of communication, such as 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), had been shown in our earlier studies to be overproduced during dual culture between the two fungal antagonists. On the other hand, the mycotoxin beauvericin was reduced in the interaction zone. The present work addresses the mechanisms underlying these changes. Hydroperoxy oxylipins are produced by lipoxygenases and P. variabile contains two lipoxygenase genes (pvlox1 and pvlox2), whereas pvlox2, but not pvlox1, is specifically up regulated during the interaction and none of the F. oxysporum lox genes vary. Heterologous expression of pvlox2 in yeast shows that the corresponding enzyme PVLOX2 produces 13-HPODE and, therefore, is most likely at the origin of the overproduced 13-HPODE during the interaction. Compellingly, beauvericin synthase gene beas expression is induced and beauvericin amounts increase in F. oxysporum mycelium when in contact with P. variabile. 13-HPODE, however, does not affect beas gene expression. Beauvericin, indeed, inhibits P. variabile growth, which counteracts and biotransforms the mycotoxin leading to reduced amounts in the interaction zone which allows further expansion of the endophyte. In order to study the interaction between the protagonists in planta, we set up an in vitro tripartite interaction assay, including the model plant Arabidopsis thaliana. F. oxysporum rapidly kills A. thaliana plants, whereas P. variabile provides up to 85% reduction of plant death if present before pathogen attack. Future studies will shed light on the protection mechanisms and the role of oxylipins and beauvericin degradation herein with the long-term aim of using endophytes in plant protection.

Phenotypic traits of Burkholderia spp. associated with ecological adaptation and plant-host interaction.

Burkholderia species have different lifestyles establishing mutualist or pathogenic associations with plants and animals. Changes in the ecological behavior of these bacteria may depend on genetic variations in response to niche adaptation. Here, we studied 15 Burkholderia strains isolated from different environments with respect to genetic and phenotypic traits. By Multilocus Sequence Analysis (MLSA) these isolates fell into 6 distinct groups. MLSA clusters did not correlate with strain antibiotic sensitivity, but with the bacterial ability to produce antimicrobial compounds and control orchid necrosis. Further, the B. seminalis strain TC3.4.2R3, a mutualistic bacterium, was inoculated into orchid plants and the interaction with the host was evaluated by analyzing the plant response and the bacterial oxidative stress response in planta. TC3.4.2R3 responded to plant colonization by increasing its own growth rate and by differential gene regulation upon oxidative stress caused by the plant, while reducing the plant's membrane lipid peroxidation. The bacterial responses to oxidative stress were recapitulated by bacterial exposure to the herbicide paraquat. We suggest that the ability of Burkholderia species to successfully establish in the rhizosphere correlates with genetic variation, whereas traits associated with antibiotic resistance are more likely to be categorized as strain specific.

Core endophyte communities of different citrus varieties from citrus growing regions in China.

The native microbiomes of citrus trees play important roles in plant health, with good communication between the native microbiome and the host plant. Here, we report on the native endophytes in 24 citrus varieties in nine citrus growing regions in China; some of the trees were healthy and others had asymptomatic or symptomatic huanglongbing, which is caused by the pathogen Candidatus Liberibacter asiaticus (CLas). We used culture-dependent analysis and characterized the isolates by partial 16S rRNA gene sequencing. The endophytes were compared between different citrus varieties, regions, and disease states (healthy, asymptomatic, and symptomatic). The total number of endophytes isolated from most of the citrus varieties was 104-106 CFU/g of leaves, but it differed significantly by disease state, with the highest numbers in the healthy leaves and the lowest in the symptomatic leaves (p < 0.05). Among the citrus varieties, the Valencia variety had the maximum number of endophyte species (22). The most dominant endophytes were Bacillus subtilis, B. velezensis, Curtobacterium luteum, and Microbacterium testaceum. The higher frequency of B. subtilis in the healthy/asymptomatic plants compared to the symptomatic plants suggests that it has a role in huanglongbing resistance. Native endophyte communities in various citrus varieties could be used to improve citrus growth and combat CLas.

Cultivation and growth dynamics of endophytic fungi in a solid culture medium based on sugar beet pulp.

BACKGROUND: The production of mycelium from endophytic fungi is of interest for applications ranging from inoculants and biofertilizers for crop production to fermentations for enzyme and metabolite production. The purpose of this study was to test the capacity of a solid growth medium based on beet pulp for growing different strains of endophytes. RESULTS: The ergosterol content of inoculated medium was measured to estimate fungal growth. Several parameters related to the preparation of the growth medium, such as water content, calcium salts and incubation time, were evaluated. The greatest fungal biomass production was observed in a medium prepared with a 1:2 (beet pulp:water) ratio, containing calcium sulfate and carbonate. Strains belonging to different fungal species grew well in the growth medium finally selected, producing yields ranging from 50 to 500 g mycelium per kilogram of dry culture, after 22-27 days. Cultures containing up to 400 g beet pulp grew successfully, and could be scaled up. CONCLUSION: A solid culture medium based on beet pulp supported the growth of diverse taxa of fungal endophytes. Both the water and calcium salt content of the growth medium affected the efficiency of mycelium production. Considering these factors, beet pulp medium was an excellent endophyte cultivation medium because of the high yield of fungal biomass observed, together with its ease of handling and scaling-up production. © 2019 Society of Chemical Industry.