Biocontrol potential of actinobacteria against Pantoea ananatis, the causal agent of maize white spot disease.

Pantoea ananatis is the causal agent of maize white spot, a foliar disease responsible for significant maize yield reduction worldwide, especially in Brazil. In general, the maize foliar diseases control involves the adoption of resistant genotypes and pesticides application. However, the use of agrochemicals can significantly cause increase production costs, damage to human health and negative environmental impacts. In this sense, the use of biological control agents has been considered among the most promising eco-friendly technologies for sustainable agriculture. Actinobacteria, particularly of Streptomyces genus, has been widely recognized as agroindustrially important microorganism due to its potential in producing diverse range of secondary metabolites, including antibiotics and enzymes. Thus, the aim of this work is to characterize and to evaluate the potential of soil actinobacteria for P. ananatis control. We observed that 59 actinobacteria strains (85%) exhibited proteolytic or chitinolytic activity. Only the strains Streptomyces pseudovenezuelae ACSL 470, that also exhibited high proteolytic activity, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 demonstrated high or moderate antagonist activity in vitro against P. ananatis. Temporal analysis of metabolites produced by these strains growth in different liquid media indicated greater antibacterial activity at 72 h. In this condition, chromatographic and mass spectrometry analysis revealed that S. pseudovenezuelae ACSL 470 strain produced neomycin, an aminoglycoside antibiotic that displayed high bactericidal activity in vitro against P. ananatis. This is the first report of actinobacteria acting as potential microbial antagonists for P. ananatis control. Further studies are needed to determine the control efficacy of maize white spot disease by Streptomyces strains or their metabolites in greenhouse and field conditions.

Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Thermo-resistant enzyme-producing microorganisms isolated from composting

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Resumo Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Expression analysis of phosphate induced genes in contrasting maize genotypes for phosphorus use efficiency.

Phosphorus is an essential nutrient for plant growth and development. The ability of plants to acquire phosphate (Pi) from the rhizosphere soil is critical in the Brazilian Cerrado characterized by acidic soil. The induction of Pi transporters is one of the earliest molecular responses to Pi deficiency in plants. In this study, we characterize the transcriptional regulation of six (ZmPT1 to ZmPT6) high-affinity Pi transporters genes in four Pi-efficient and four Pi-inefficient maize (Zea mays) genotypes. The expression analysis indicated that Pi-starvation induced the transcription of all ZmPT genes tested. The abundance of transcripts was inversely related to Pi concentration in nutrient solution and was observed as early as five days following the Pi deprivation. The Pi-starved plants replenished with 250 µM Pi for four to five days resulted in ZmPT suppression, indicating the Pi role in gene expression. The tissue-specific expression analysis revealed the abundance of ZmPT transcripts in roots and shoots. The six maize Pi transporters were primarily detected in the upper and middle root portions and barely expressed in root tips. The expression profiles of the six ZmPTs phosphate transporters between and among Pi-efficient and Pi-inefficient genotypes showed an absence of significant differences in the expression pattern of the ZmPTs among Pi-efficient and Pi-inefficient genotypes. The results suggested that Pi acquisition efficiency is a complex trait determined by quantitative loci in maize.

Resistance of sorghum hybrids to sorghum aphid.

The aphid Melanaphis sorghi (Theobald) (Hemiptera: Aphididae), which infest the sorghum crop, has been an economically important pest which have been causing severe damage to sorghum crops in Brazil since 2019. These species have been observed mainly at the end of vegetative stage and beginning of reproductive stage of plants. Their high reproductive rate on sorghum raises concerns about these pests. Therefore, the present study aimed to estimate the life expectancy and fertility tables of Melanaphis sorghi fed on 15 hybrids of grain sorghum, in order to know the resistance characteristics of these materials and understand how plant resistance can help manage this insect. This study was carried out in a laboratory at 26±2 ºC and 60±10% R.H (relative humidity). Fifty insects were kept in each hybrid, each insect was considered one repetition. According to biological parameters and fertility life table, hybrids BRS373, DKB590 and 50A10 were less suitable for the sugarcane aphid multiplication. Thus, these hybrids could be useful to manage this pest, since the population growth speed is one of the worst problems for the implementation of cropping systems. Genotypes AG1090, MSK327 and XGN1305 favored the development of this pest and, when chosen within a cropping system, other management strategies should be considered.

Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides.

The mycotoxigenic fungus Fusarium verticillioides is the primary maize pathogen and causes the maize stalk and ear rot diseases with significant economic losses. Furthermore, the excessive use of fungicides to control F. verticillioides constitutes threats to the environment and human health. Thus, sustainable alternatives such as biological control are needed to minimize the hazards associated with the current method. Although much is known about the vulnerability of the maize silks as a gateway for several fungal pathogens invading the developing grains, studies on the chemical properties of silk extracts and their resident microbiota are scarce. This study isolated and characterized bacteria and fungi that colonize the maize stigma to assess new potential biocontrol agents. The samples were collected from maize fields in the Brazilian localities of Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR, and Goiânia-GO. One hundred sixty-seven microorganisms were isolated, 46% endophytic and 54% epiphytic. First, the antagonist activity was evaluated by the agar disc diffusion method performed in triplicate, and 83% of the isolates showed antagonist activity against F. verticillioides. Then, the 42 most efficient isolates were identified based on the partial sequencing of the bacterial 16S rRNA gene and fungi ITS region. The bacteria belong to the genera Bacillus (57.1%), Burkholderia (23.8%), Achromobacter (7.1%), Pseudomonas (2.4%), and Serratia (2.4%), while the fungi are Penicillium (2.4%), Candida (2.4), and Aspergillus (2.4%). The results showed that microorganisms from maize stigma might represent new promising agents for F. verticillioides control.

Thermo-resistant enzyme-producing microorganisms isolated from composting.

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Characterization and phylogenetic affiliation of Actinobacteria from tropical soils with potential uses for agro-industrial processes.

Secondary metabolites produced by Actinobacteria of tropical soils represent a largely understudied source of novel molecules with relevant application in medicine, pharmaceutical and food industries, agriculture, and environmental bioremediation. The present study aimed to characterize sixty-nine Actinobacteria isolated from compost and tropical soils using morphological, biochemical, and molecular methods. All the isolates showed high variation for morphological traits considering the color of pigments of the aerial and vegetative mycelium and spore chain morphology. The enzymatic activity of amylase, cellulase, and lipase was highly variable. The amylase activity was detected in 53 (76.81%) isolates. Eighteen isolates showed enzymatic index (EI) > 4.0, and the isolates ACJ 45 (Streptomyces curacoi) and ACSL 6 (S. hygroscopicus) showed the highest EI values (6.44 and 6.42, respectively). The cellulase activity varied significantly (P ≤ 0.05) among the isolates. Twenty-nine isolates (42.02%) showed high cellulase activity, and the isolates ACJ 48 (S. chiangmaiensis) and ACJ 53 (S. cyslabdanicus) showed the highest EI values (6.56 for both isolates). The lipase activity varied statistically (P ≤ 0.05) with fourteen isolates (20.29%) considered good lipase producers (EI > 2.0). The isolate ACSL 6 (S. hygroscopicus) showed the highest EI value of 2.60. Molecular analysis of partial 16S rRNA gene sequencing revealed the existence of 49 species, being 38 species with only one representative member and 11 species represented by one or more strains. All species belonged to three genera, namely Streptomyces (82.61%), Amycolatopsis (7.25%), and Kitasatospora (10.14%). The present results showed the high biotechnological potential of different Actinobacteria from tropical soils.

Maize endophytic bacteria as mineral phosphate solubilizers.

In the present study, we demonstrated the in vitro activity of endophytic phosphate-solubilizing bacteria (PSB). Fifty-five endophytic PSB that were isolated from sap, leaves, and roots of maize were tested for their ability to solubilize tricalcium phosphate and produce organic acid. Partial sequencing of the 16S rRNA-encoding gene showed that the isolates were from the genus Bacillus and different species of Enterobacteriaceae. The phosphate solubilization index on solid medium and phosphate solubilization in liquid medium varied significantly among the isolates. There was a statistically significant difference (P ≤ 0.05) for both, the values of phosphate-solubilizing activity and pH of the growth medium, among the isolates. Pearson correlation was statistically significant (P ≤ 0.05) between P-solubilization and pH (R = -0.38), and between the gluconic acid production and the lowering of the pH of the liquid medium at 6 (R = 0.28) and 9 days (R = 0.39). Gluconic acid production was prevalent in all the PSB studied, and Bacillus species were most efficient in solubilizing phosphate. This is the first report on the characterization of bacterial endophytes from maize and their use as potential biofertilizers. In addition, this may provide an alternative strategy for improving the phosphorus acquisition efficiency of crop plants in tropical soils.

Molecular detection of nifH gene-containing Paenibacillus in the rhizosphere of sorghum (Sorghum bicolor) sown in Cerrado soil.

AIMS: To develop a polymerase chain reaction (PCR)-based approach for the detection of nifH gene-containing Paenibacillus in environmental samples. METHODS AND RESULTS: The primers, nifHPAENf and nifHPAENr, were designed and tested with DNA from: (i) strains of different nitrogen-fixing Paenibacillus species, (ii) strains of other nitrogen-fixing genera and (iii) rhizosphere of sorghum sown in Cerrado soil amended with either 12 or 120 kg ha(-1) of nitrogen fertilizer. All nitrogen-fixing Paenibacillus strains tested and the DNA samples from rhizosphere soil were amplified when these primers were used, generating a 280 bp fragment. When the PCR products obtained from both sorghum rhizospheres were cloned and sequenced, the majority of the clones analysed could be identified as Paenibacillus durus. Moreover, a greater diversity in the nifH sequences could be observed in the rhizosphere treated with a high amount of nitrogen fertilizer. CONCLUSIONS: Nitrogen fertilization slightly influenced the structure of the nifH gene-containing Paenibacillus community in sorghum rhizospheres cultivated in Cerrado soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR detection method developed is adequate to assess the presence of nifH gene-containing Paenibacillus in the environment and can be used in future to determine the ecological role of this group of micro-organisms for the nitrogen input to the plants.

Ecological distribution of Spirillum lipoferum Beijerinck.

A survey in various countries revealed that the N2-fixing Spirillum lipoferum Beijerinck is a very common root and soil inhabitant in the tropics. More than half of the grass root and soil samples collected in tropical countries (four African countries and Brazil) contained abundant S. lipoferum populations, while less than 10% of the samples collected in temperate South Brazil, Kenya, and the U.S.A. contained the organism. There is a pronounced vegetation effect. Panicum maximum seems the most favorable among the forage grasses, while few positive samples were found under virgin tropical forest. Legume roots contained less S. lipoferum than adjacent soils. More than 80% of the samples from cereal roots (maize, sorghum, wheat, and rye) grown in fields fertilized with PK and Mo, in Rio de Janeiro State, were positive. Maize and sorghum grown under similar conditions in Wisconsin contained less than 10% of positive samples, but when maize fields were inoculated 90% of the root samples contained S. lipoferum. Alluvial soils were more favorable than eroded hill soils. Occurrence in soil was strongly pH-dependent with a pH around 7, being optimal (correlation coefficient r = 0.90). Sporadic occurrence was observed even in soils with pH 4.8. Surface-sterilized P. maximum roots collected from soils with pH ranging from 4.8 to 7.2 contained high S. lipoferum numbers which did not correlate with soil pH (r = 0.41). Amendment with malate of acid soils was not very effective in increasing nitrogenase (N2-ase) activity, but in two soils with pH above 6.4, high N2-ase activity was obtained after 16 to 48 h of incubation. In two soils from a temperate climate region, inoculation with S. lipoferum increased N2-ase activity produced through malate amendment.