Effective plant-endophyte interplay can improve the cadmium hyperaccumulation in Brachiaria mutica.

Soil contamination due to cadmium (Cd) is a ubiquitous environmental problem for which inexpensive remediation alternatives are required. Phytoaccumulation, the use of plants to extract and accumulate heavy metals from the contaminated environment, is such an alternative. In this study, we aimed at establishing effective plant-bacteria interplay between Brachiaria mutica and Cd-resistant endophytic bacteria eventually leading to improved phytoremediation. B. mutica was grown in a Cd-contaminated soil and inoculated with three Cd-tolerant endophytic bacteria individually as well as in combination. Plant physiological parameters, biomass production, bacterial colonization, and Cd-accumulation were observed at four different Cd exposures, i.e., 100, 200, 400 and 1000 mg kg-1 of soil. The combined application of endophytic bacteria was more effective as compared to their individual applications at all concentrations. Nevertheless, highest performance of consortium was seen at 100 mg Cd kg-1 of soil, i.e., root length was enhanced by 46%, shoot length by 62%, chlorophyll content by 40%, and dry biomass by 64%; which was reduced with the increase in Cd concentration. The bacterial population was highest in the root interior followed by rhizosphere and shoot interior. Concomitantly, plants inoculated with bacterial consortium displayed more Cd-accumulation in the roots (95%), shoots (55%), and leaves (44%). Higher values of BCFroot (> 1), and lower values for BCFshoot and TF (< 1) indicates capability of B. mutica to accumulate high amounts of Cd in the roots as compared to the aerial parts. The present study concludes that plant-endophyte interplay could be a sustainable and effective strategy for Cd removal from the contaminated soils.

Plant growth promoting bacteria in Brachiaria brizantha.

Brachiaria brizantha is considered one of the preferred fodders among farmers for having high forage yield and large production of root mass. The association of beneficial bacteria with these grasses can be very valuable in the recovery of the pasture areas with nutritional deficiency. With the aim of studying this possibility, we carried out the sampling of soil and roots of B. brizantha in three areas (Nova Odessa-SP, São Carlos-SP and Campo Verde-MT, Brazil). Seventy-two bacterial strains were isolated and used in tests to evaluate their biotechnological potential. Almost all isolates presented at least one positive feature. Sixty-eight isolates produced analogues of indole-3-acetic acid, ten showed nitrogenase activity when subjected to the method of increasing the concentration of total nitrogen (total N) in the culture medium and sixty-five isolates showed nitrogenase activity when subjected to acetylene reduction technique. The partial sequencing of 16S rRNA of these isolates allowed the identification of seven main groups, with the prevalence of those affiliated to the genus Stenotrophomonas (69 %). At the end, this work elected the strains C4 (Pseudomonadaceae) and C7 (Rhodospirillaceae) as promising organisms for the development of inoculants due to their higher nitrogenase activity.

Experimental validation under controlled conditions of real time PCR to quantify arbuscular mycorrhizal colonization in root.

Mycorrhizal colonization of roots is traditionally evaluated by empirical methods, such as root microscopy. We compared this method with data from using a real time PCR technique, and determined the correlation between methods, indicating particularities of a promising system for a quick and accurate molecular diagnostic of arbuscular mycorrhization.

Effect of physical, chemical and environmental characteristics on arbuscular mycorrhizal fungi in Brachiaria decumbens (Stapf) pastures.

AIM: To evaluate the effects of soil physical and chemical factors (pH, conductivity, humidity, available phosphorus and organic matter) and environmental factors (temperature, relative air humidity, altitude and atmospheric pressure) on arbuscular mycorrhizal fungi (AMF)-Brachiaria decumbens grass relationship. Furthermore to establish patterns of microbiological responses that allow to differentiate the study sites in two relief types. METHODS AND RESULTS: Mycorrhizal characteristics (spore density, external hyphae and root colonizations by hyphae, vesicles and arbuscules), physical and chemical factors in soil and environmental factors were measured. CONCLUSIONS: The effect of physical, chemical and environmental factors on microbiological variables was related to the type of relief 'valley and hilly terrain'; the AMF behaviour was affected only over narrower ranges of evaluated variables. Similarly, the colonization of B. decumbens roots by AMF hyphae, vesicles and the mycorrhizal spore density follow different patterns according to the relief type. SIGNIFICANCE AND IMPACT OF THE STUDY: The type of relief is one of the factors to be taken into consideration to evaluate the AMF inoculum and root colonization of these pastures, because of the influence of slope - as physical property of soil - on AMF.

Pleurotus spp. cultivation on Brachiaria sp. straw treatment with alkaline water: Oyster mushroom and substrate treatment.

The aim of this research was to evaluate the efficiency of aqueous alkali-treated Brachiaria straw for the cultivation of appropriate species of oyster mushroom. The substrate used in the cultivation of various Pleurotus spp. was soaked for 20min by using two different procedures: (i) 0.5-2.0% Ca(OH)2 in 100L water, and (ii) 50-250L water. As a result, 1% Ca(OH)2 dissolved in 100L water and 3.5kg of Brachiaria straw presented the best production. The most suitable species for the application of the present method were P. pulmonarius and P. sapidus. The success of this technique is directly related to the concentration of Ca(OH)2 and water, the species, and the origin and quality of raw material used as the substrate in the production of oyster mushroom.

Brachiaria Grasses (Brachiaria spp.) harbor a diverse bacterial community with multiple attributes beneficial to plant growth and development.

Endophytic and plant-associated bacteria were isolated from plants and rhizoplane soil of naturally grown Brachiaria grasses at International Livestock Research Institute in Nairobi, Kenya. Eighty-four bacterial strains were isolated from leaf tissues, root tissues, and rhizoplane soil on nutrient agar and 869 media. All bacterial strains were identified to the lowest possible taxonomic unit using 16S rDNA primers and were characterized for the production of Indole-3-acetic acid, hydrogen cyanide, and ACC deaminase; phosphate solubilization; siderophore production; antifungal properties; and plant biomass production. The 16S rDNA-based identification grouped these 84 bacterial strains into 3 phyla, 5 classes, 8 orders, 12 families, 16 genera, and 50 unique taxa. The four most frequently isolated genera were Pseudomonas (23), Pantoea (17), Acinetobacter (9), and Enterobacter (8). The functional characterization of these strains revealed that 41 of 84 strains had a minimum of three plant beneficial properties. Inoculation of maize seedlings with Acinetobacter spp., Microbacterium spp., Pectobacterium spp., Pseudomonas spp., and Enterobacter spp. showed positive effects on seedling biomass production. The ability of Brachiaria grasses to host genetically diverse bacteria, many of them with multiple plant growth-promoting attributes, might have contributed to high biomass production and adaptation of Brachiaria grasses to drought and low fertility soils.

Pleurotus spp. cultivation on Brachiaria sp. straw treatment with alkaline water

Abstract The aim of this research was to evaluate the efficiency of aqueous alkali-treated Brachiaria straw for the cultivation of appropriate species of oyster mushroom. The substrate used in the cultivation of various Pleurotus spp. was soaked for 20 min by using two different procedures: (i) 0.5-2.0% Ca(OH)2 in 100 L water, and (ii) 50-250 L water. As a result, 1% Ca(OH)2 dissolved in 100 L water and 3.5 kg of Brachiaria straw presented the best production. The most suitable species for the application of the present method were P. pulmonarius and P. sapidus. The success of this technique is directly related to the concentration of Ca(OH)2 and water, the species, and the origin and quality of raw material used as the substrate in the production of oyster mushroom.

Agrobacterium-mediated transformation of the endophytic fungus Acremonium implicatum associated with Brachiaria grasses.

Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophyte's plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties.

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.