Evaluation of the benefits of plant growth-promoting rhizobacteria and mycorrhizal fungi on biochemical and morphophysiological traits of Aloe barbadensis Mill under water deficit stress.

Aloe barbadensis is a drought-tolerant perennial medicinal plant with both nutritional and cosmetic uses. Drought is one of the main abiotic stresses limiting plant growth and development. However, the use of drought-resistant plants combined with beneficial soil micro-organisms could improve the effectiveness of biological methods to mitigate drought damage. This research aims to evaluate the effects of Funneliformis mosseae (MF), plant growth-promoting rhizobacteria (PGPR) (including Pseudomonas putida and Pantoea agglomerans), and their co-inoculation on the macronutrient status, antioxidant enzyme activities, and other morphophysiological traits of A. barbadensis under four irrigation regimes [25%, 50%, 75% and 100% of water requirement (WR)]. Three harvests were conducted, revealing that inoculation enhanced the survival rate and shoot fresh weight (SFW) compared to the control plants. However, at 25% WR, the SFW was reduced by 43% more than the control. across all harvests, while the PGPR + MF treatment showed increases of more than 19%, 11%, and 17% compared to the control, MF, and PGPR treatments, respectively. The results also showed that A. barbadensis exhibited innate drought tolerance up to a 50% WR level by enhancing physiological defenses, such as antioxidant enzyme activity. Inoculation increased the macronutrient status of the plant at all levels of irrigation regimes especially under severe drought conditions. The highest levels of nitrogen (N) (16.24 mg g-1 DW) and phosphorus (P) (11.29 mg g-1 DW) were observed in the PGPR + MF treatment at 100% WR. The maximum relative water content under MF inoculation and 75% WR (98.24%) (98.24%) was reached. PGPR + MF treatment alleviated drought-induced osmotic stress, as indicated by reduced antioxidant enzyme activities and electrolyte leakage. However, P. putida and P. agglomerans strains alone or in combination with F. mosseae increased plant yield, macronutrient uptake and antioxidant enzyme activity. This study underscores the potential of these PGPR and MF strains as invaluable biological tools for the cultivation of A. barbadensis in regions with severe drought stress.

Isolation, identification and bioactivity analysis of an endophytic fungus isolated from Aloe vera collected from Asir desert, Saudi Arabia.

Endophytic fungi isolated from desert plants are among the less known organisms with potentially valuable applications. The bioactivities of an endophytic fungus isolated from Aloe vera, a plant found in central regions of Asir desert, Saudi Arabia. Based on primary phytochemical screening, an efficient isolate was selected and identified according to the sequence analysis of the internal spacer regions ITS1, ITS4 and the 5.8S region as Preussia africana belonging to the family Sporormiaceae. The crude extract of this fungus was evaluated for its bioactivities. Under static conditions, the crude extract at a concentration of 500 µg/mL had a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging rate of 87%, whereas a higher concentration (100 µg/mL) had an astounding wound healing effect (42.6% at 48 h) when compared to positive control. Moreover, the crude extract with a concentration of 50 µg/mL was active against almost all cancer cell lines such as HeLa (cervical cancer), Hep G2 (liver cancer), MCF-7 (breast cancer), A549 (lung cancer), LN-229 (glioblastoma), A-431 (skin cancer), and kidney cell line (HEK 293T). The results suggest that the endophytic fungus P. africana from A. vera has wide therapeutic applications against severe disease conditions.

Actinomycin-Producing Endophytic Streptomyces parvulus Associated with Root of Aloe vera and Optimization of Conditions for Antibiotic Production.

Endophytic actinomycetes are a rich source of novel antimicrobial compounds. The aim of this study was to evaluate the production of antimicrobial compound by endophytic Streptomyces sp. Av-R5 associated with root of Aloe vera against multidrug-resistant human pathogens. The 16S rRNA sequence of the isolate Av-R5 has been identified as Streptomyces parvulus NBRC 13193T (AB184326) and the sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank database (accession number KY771080). Streptomyces parvulus Av-R5 grown under submerged fermentation condition optimized by central composite design (glucose 11.16 g/L, soybean meal 10.25 g/L, sodium chloride 11.18 g/L, calcium carbonate 1.32 g/L at pH 7.19 at 31.42 °C with 6.04% seed inoculum for 10 days of incubation) exhibited the highest activity against multidrug-resistant Staphylococcus aureus JNMC-3, Staphylococcus epidermidis JNMC-4, Klebsiella pneumoniae MTCC-3384, Klebsiella pneumoniae JNMC-6, Pseudomonas aeruginosa MTCC-741, Proteus vulgaris JNMC-7, Candida albicans MTCC-183, and Aspergillus niger MTCC-872. The structures of the active compounds were elucidated by UV-Vis spectroscopy, 1H and 13C NMR, FT-IR, and ESIMS. Actinomycin D and actinomycin X0ß were detected in crude extracts and major components were eluted by HPLC at 10.96 and 6.81 min, respectively. In this case, a high yield of actinomycin D and actinomycin X0ß (400 mg/L) was achieved with Streptomyces parvulus Av-R5, fermented in glucose soybean meal broth media, which can be used in industrial fermentation process to obtain high yields.

Encountering epidemic effects of leaf spot disease (Alternaria brassicae) on Aloe vera by fungal biocontrol agents in agrifields-An ecofriendly approach.

Aloe vera (L.) Burm.f. is a highly important and extensively cultivated medicinal plant and that is also extensively used in the cosmetic industry. It has been frequently reported to suffer from Alternaria leaf spot disease in various parts of the world. Various fungicides used to combat this disease, have deleterious effects on the environment and on pharmacologically important constituents of Aloe vera. To avoid the harmful effects of fungicides an ecofriendly approach has been adopted here. A weekly survey was conducted during 2013-2015 in and around North 24 Parganas (West Bengal) to obtain the percentage of disease index (PDI). For biological control of the disease, screening of the antagonistic efficacy of biocontrol agents was carried out through the in vitro dual-culture-plate method and scanning electron microscopy (SEM) was used to study the mechanism. The in vitro effects of fungicides on the radial growth of the pathogen were evaluated through the poison food method and were compared with potent antagonistic fungi. Field application of potent antagonistic fungi was conducted through the dip-and-spray method. The results showed that, the PDI peaked during the hot and humid conditions of May to September (76.57%-98.57%) but decreased during the winter, December-January (35.71-46.66%). Trichoderma asperellum exerted the greatest inhibition of the radial growth of A. brassicae acting through non volatile (70.39%) and volatile metabolites (72.17%). A SEM study confirmed the hyperparasitic nature of T. asperellum through hyphal coiling-T. asperellum was similar to 2% blitox-50 (73.92%) and better than 2% bavistin (59.77%) (in vitro). In agricultural field trials (2013-15), Trichoderma application restricted the disease to the smallest area (PDI 24.00-29.33%) in comparison to untreated plots (73.33%). In conclusion, saplings treated with the dip method (108 spores / mL) and sprayed 4 times with a spore suspension of biocontrol agents such as T. asperellum, T. viride and T. harzianum, standardized at a rate of 2.5 L / plot (36 sq ft) (108 spores/ mL) are suggested for the ecofriendly management of this epidemic leaf spot disease of Aloe vera in agricultural fields.

Achromobacter aloeverae sp. nov., isolated from the root of Aloe vera (L.) Burm.f.

Two Gram-staining-negative, strictly aerobic, rod-shaped bacteria, designated strains AVA-1T and AVA-2, were isolated from the root of Aloe vera (L.) Brum.f. derived from Chachoengsao Province, Thailand. The strains contained cytochrome oxidase and catalase activities. They grew in 4 % (w/v) NaCl, at a pH range of 6.0-9.0 (optimally at pH 7) and at 20-42 °C (optimally at 30-37 °C). The major isoprenoid quinone was ubiquinone with eight isoprene units (Q-8). The major fatty acids were C16 : 0 and C17 : 0 cyclo. On the basis of 16S rRNA gene sequence analysis, the strains represent a species belonging to the genus Achromobacter and are closely related to Achromobacter xylosoxidans NBRC 15126T (98.80 %), Achromobacter insolitus LMG 6003T (98.64 %), Achromobacter aminicus LMG 26690T (98.59 %), Achromobacter pulmonis LMG 26696T (98.58 %) and Achromobacter insuavis LMG 26845T (98.58 %). The DNA G+C content of strain AVA-1T was 66.5 mol%. The novel strains had low DNA-DNA relatedness values with related type strains. On the basis of the phenotypic and genotypic data obtained, the strains clearly represent a novel species, for which the name Achromobacter aloeverae sp. nov. is proposed. The type strain is strain AVA-1T (=LMG 29108T=NBRC 111463T=PCU 352T=TISTR 2383T).

Diversity, antimicrobial and antioxidant activities of culturable bacterial endophyte communities in Aloe vera.

Twenty-nine culturable bacterial endophytes were isolated from surface-sterilized tissues (root, stem and leaf) of Aloe vera and molecularly characterized to 13 genera: Pseudomonas, Bacillus, Enterobacter, Pantoea, Chryseobacterium, Sphingobacterium, Aeromonas, Providencia, Cedecea, Klebsiella, Cronobacter, Macrococcus and Shigella. The dominant genera include Bacillus (20.7%), Pseudomonas (20.7%) and Enterobacter (13.8%). The crude and ethyl acetate fractions of the metabolites of six isolates, species of Pseudomonas, Bacillus, Chryseobacterium and Shigella, have broad spectral antimicrobial activities against pathogenic Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes and Candida albicans, with inhibition zones ranging from 6.0 ± 0.57 to 16.6 ± 0.57 mm. In addition, 80% of the bacterial endophytes produced 1,1-diphenyl-2-picrylhydrazyl (DPPH) with scavenging properties of over 75% when their crude metabolites were compared with ascorbic acid (92%). In conclusion, this study revealed for the first time the endophytic bacteria communities from A. vera (Pseudomonas hibiscicola, Macrococcus caseolyticus, Enterobacter ludwigii, Bacillus anthracis) that produce bioactive compounds with high DPPH scavenging properties (75-88%) and (Bacillus tequilensis, Pseudomonas entomophila, Chryseobacterium indologenes, Bacillus aerophilus) that produce bioactive compounds with antimicrobial activities against bacterial pathogens. Hence, we suggest further investigation and characterization of their bioactive compounds.

Characterization of in vitro antifungal activities of small and American cranberry (Vaccinium oxycoccos L. and V. macrocarpon Aiton) and lingonberry (Vaccinium vitis-idaea L.) concentrates in sugar reduced fruit spreads.

In this study, cranberry and lingonberry concentrates were added to commercial sugar-reduced fruit spreads (raspberry-Aloe vera, strawberry-guava, and strawberry-lime), and tested for their antifungal activities. Selected strains of the species Absidia glauca, Penicillium brevicompactum, Saccharomyces cerevisiae and Zygosaccharomyces bailii, as well as xerophilic environmental isolates of the genera Penicillium and Eurotium were used for challenge testing. Initially, varying concentrations of synthetic antifungal agents, such as sodium benzoate, potassium sorbate and butyl 4-hydroxybenzoate were tested against these fungi on wort agar containing 31% fructose at different pH values. Subsequently, the experiments were conducted in fruit spreads containing different concentrations of cranberry and lingonberry concentrates. The results of this study demonstrate that these concentrates were able to inhibit growth of visible colonies of xerophilic and non-xerophilic fungi. Cranberry and lingonberry concentrates are interesting candidates for natural preservation against fungal growth in sugar reduced fruit spreads.

Description of Micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from Aloe vera.

A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6(T), was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6(T) grew at high salt concentrations [10% (w/v) NaCl], temperatures of 15-41 °C and a pH range of 5-12. It showed highest (99.7%) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004(T) followed by Micrococcus luteus NCTC 2665(T) (99.6%) and Micrococcus endophyticus YIM 56238(T) (99.0%). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004(T) and M. luteus NCTC 2665(T). Like other members of the genus Micrococcus, strain AE-6(T) had a high content of branched chain fatty acids (iso-C15:0 and anteiso-C15:0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an 'A2 L-Lys-peptide subunit' type of peptidoglycan and ribose to be the major cell wall sugar. The DNA G+C content was 70 mol%. Results of DNA-DNA hybridization of AE-6(T) with its closest relatives from the genus Micrococcus produced a value of less than 70%. Based on the results of this study, strain AE-6(T) could be clearly differentiated from other members of the genus Micrococcus. We propose that it represents a novel species of the genus Micrococcus and suggest the name Micrococcus aloeverae sp. nov., with strain AE-6(T) ( = MCC 2184(T) = DSM 27472(T)) as the type strain of the species.

[Inhibition effects and mechanisms of the entophytic fungus Trichoderma harzianum LH-7 from Aloe barbadensis].

Inhibition spectrum and antagonistic mechanism of an endophytic fungus Trichoderma harzianum LH-7, isolated from wild medicinal plant Aloe barbadensis, were investigated by in vitro culture methods against 9 kinds of plant pathogens. The results showed that nutrient competition and hyper-parasitism were the two primarily antagonist approaches that strain LH-7 adopted to inhibit the tested plant pathogens with a significant inhibition rate of 62.4%-88.4%. Moreover, the active compound from metabolites of LH-7 could cause pathogen mycelial deformities, cell wall rupture and conidial malformation, leading to the effective inhibition on pathogens growth and reproduction.

Lactobacillus brevis strains from fermented aloe vera survive gastroduodenal environment and suppress common food borne enteropathogens.

Five novel Lactobacillus brevis strains were isolated from naturally fermented Aloe vera leaf flesh. Each strain was identified by Random Amplified Polymorphic DNA (RAPD) analysis and 16S rRNA sequence comparison. These strains were highly tolerant to acid, surviving in pH2.5 for up to 4 hours, and resistant to 5% bile salts at 37°C for 18 hours. Due to its tolerance to acid and bile salts, one strain passed through the gastric barrier and colonised the intestine after oral administration. All five strains inhibited the growth of many harmful enteropathogens without restraining most of normal commensals in the gut and hence named POAL (Probiotics Originating from Aloe Leaf) strains. Additionally, each strain exhibited discriminative resistance to a wide range of antibiotics. The L. brevis POAL strains, moreover, expressed high levels of the glutamate decarboxylase (GAD) gene which produces a beneficial neurotransmitter, γ-aminobutyric acid (GABA). These characteristics in all suggest that the novel L. brevis strains should be considered as potential food additives and resources for pharmaceutical research.

Streptomyces zhaozhouensis sp. nov., an actinomycete isolated from candelabra aloe (Aloe arborescens Mill).

A novel endophytic actinomycete, designated strain NEAU-LZS-5(T), was isolated from the leaf of candelabra aloe (Aloe arborescens Mill) and characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence showed that strain NEAU-LZS-5(T) belongs to the genus Streptomyces and exhibited 99.51 and 97.37 % similarity to Streptomyces sedi YIM 65188(T) and Streptomyces specialis GW41-1564(T), respectively, whereas low similarity values (<97 %) distinguished strain NEAU-LZS-5(T) from all other species of the genus Streptomyces with validly published names. Two tree-making algorithms also supported the position that strain NEAU-LZS-5(T) formed a distinct clade with Streptomyces sedi YIM 65188(T) and Streptomyces specialis GW41-1564(T). However, levels of DNA-DNA relatedness between strain NEAU-LZS-5(T) and Streptomyces sedi YIM 65188(T) and Streptomyces specialis GW41-1564(T) were 45.59 and 31.90 %, respectively. A comparative study between strain NEAU-LZS-5(T) and the type strains of closest related species of the genus Streptomyces revealed that it differed from them in morphological, physiological and biochemical characteristics. Therefore, strain NEAU-LZS-5(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces zhaozhouensis sp. nov. is proposed. The type strain is NEAU-LZS-5(T) ( = CGMCC 4.7095(T) = DSM 42101(T)).

Atropisomeric dihydroanthracenones as inhibitors of multiresistant Staphylococcus aureus.

Two bisdihydroanthracenone atropodiastereomeric pairs, including homodimeric flavomannin A (1) and the previously unreported flavomannin B (2), two new unsymmetrical dimers (3 and 4), and two new mixed dihydroanthracenone/anthraquinone dimers (5 and 6) were isolated from Talaromyces wortmannii , an endophyte of Aloe vera . The structures of 2-6 were elucidated by extensive NMR and mass spectrometric analyses. The axial chirality of the biaryls was determined using TDDFT ECD and VCD calculations, the combination of which however did not allow the assignment of the central chirality elements of 1. The compounds exhibited antibacterial activity against Staphylococcus aureus , including (multi)drug-resistant clinical isolates. Reporter gene analyses indicated induction of the SOS response for some of the derivatives, suggesting interference with DNA structure or metabolism. Fluorescence microscopy demonstrated defective segregation of the bacterial chromosome and DNA degradation. Notably, the compounds showed no cytotoxic activity, encouraging their further evaluation as potential starting points for antibacterial drug development.

Microbiological stabilization of Aloe vera (Aloe barbadensis Miller) gel by high hydrostatic pressure treatment.

The effect of high hydrostatic pressure (HHP) treatment (300, 400 and 500 MPa for 1 and 3 min at 20 °C) on the microbiological shelf-life and microbiota composition of Aloe vera gel during 90 days of storage at 4 °C was investigated. Aerobic mesophilic and psychrotrophic bacteria, as well as moulds and yeasts, were enumerated after HHP treatment and through cold storage. Randomly selected isolates from the count plates were identified by standard methods and the API identification system. Results showed that HHP treatment at or over 400 MPa for 3 min were effective to keep the microbial counts to undetectable levels during the whole storage period, and consequently the microbiological shelf-life of A. vera gel was extended for more than 90 days at 4 °C. The microbiota in the untreated A. vera gel was dominated by Gram-negative bacteria (mostly Rahnella aquatilis) and yeasts (mostly Rhodotorula mucilaginosa). In contrast, Gram-positive bacteria tentatively identified as Arthrobacter spp. and Micrococcus/Kocuria spp. were the predominant microorganisms in samples pressurized at 300 MPa for 1 and 3 min, while Bacillus megaterium predominating in samples treated at 400 MPa for 1 min. At 400 MPa for 3 min and above, the microbial growth was completely suppressed during at least 90 days; however, viable spore-formers were detected by enrichment.

Isolation and identification of phosphate solubilizing bacteria able to enhance the growth and aloin-A biosynthesis of Aloe barbadensis Miller.

The effect of four phosphate solubilizing bacteria (PSB) was studied on growth and aloin-A content of Aloe barbadensis in soil containing tricalcium phosphate (TCP). PSB were identified based on 16S rRNA gene sequencing as Pseudomonas synxantha, Burkholderia gladioli, Enterobacter hormaechei and Serratia marcescens. These PSB solubilized 25-340 µg ml(-1) of TCP into the liquid phase. The treatment of plants with individual PSB or mixture of these increased soil available P, P uptake in plants and plant growth. The increase in aloin-A content due to higher plant biomass and unit biomass production was 673%, 294%, 276%, 119% and 108% in plants treated with a PSB consortium, P. synxantha, S. marcescens, B. gladioli, and E. hormaechei in TCP amended soil, respectively.

Studies on rhizosphere microbial diversity of some commercially important medicinal plants.

A preliminary study was made on four medicinal plants viz., Ocimum sanctum L., Coleus forskholii Briq, Catharanthus roseus (L.) G. Don. and Aloe vera in order to identify and enumerate the rhizosphere, non-rhizosphere and diazotrophic microorganisms in soil. The diazotrophic bacterial population studied includes Azospirillum, Azotobacter and Pseudomonas. The rhizosphere bacterial populations were 23.33 x 10(6)g(-1) in O. sanctum followed by C. roseus (20.46 x 10(6)g(-1)), A. vera (18.44 x 10(6)g(-1)) and C. forskholii (16.64 x 10(6)g(-1)). The fungi populations were 19.44 x 10(4)g(-1) in C. roseus, 18.66 x 10(4)g(-1) in O. sanctum, 16.44 x 10(4)g(-1) in A. vera and 14.22 x 10(4)g(-1) in C. forskholii. The actinomycetes population was 12.22 x 10(5)g(-1) in O. sanctum, 10.44 x 10(5)g(-1) in C. roseus, 8.44 x 10(5)g(-1) in A. vera and 6.22 x 10(5)g(-1) in C. forskholii. The diazotrophic bacterial population of Azospirillum, Azotobacter and Pseudomonas is 8.2 x 10(4)g(-1), 12 x 10(4)g(-1), 6 x 10(4)g(-1) in the rhizosphere soil. In all the four medicinal plants the microbial population is more in the rhizosphere soil, when compared to non-rhizosphere soil. These results are helpful in developing a biofertilizer consortium for these commercially grown medicinal plants.

[Study on application of arbuscular-mycorrhizas in growing seedings of Aloe vera].

Tissue culture seedlings of Aloe vera L. inoculated with 7 AMF(arbuscular mycorrhiza fungi) in a greenhouse in Guangzhou showed that the percentage of infection was 99.67%-100%, the index of infected was 73.3%-86.67%. After being inoculated 13 months, the seedling high raised 19.88%-51.91%, the leaves length raised 13.13%-150.96%. After being inoculated 15 months, the leaves juice of Aloe vera raised 60.87%-233.8% and the dried of leaves juice raised 217%-724%.