Walter Maccheroni

Endophytic microorganisms: a review on insect control and recent advances on tropical plants

In the past two decades, a great deal of information on the role of endophytic microorganisms in nature has been collected. The capability of colonizing internal host tissues has made endophytes valuable for agriculture as a tool to improve crop performance. In this review, we addressed the major topics concerning the control of insects-pests by endophytic microorganisms. Several examples of insect control are described, notably those involving the interactions between fungi and grazing grasses from temperate countries. The mechanisms by which endophytic fungi control insect attacks are listed and include toxin production as well as the influence of these compounds on plant and livestock and how their production may be affected by genetic and environmental conditions. The importance of endophytic entomopathogenic fungi for insect control is also addressed. As the literature has shown, there is a lack of information on endophytes from tropical hosts, which are more severely affected by pests and diseases. Having this in mind, we have included an updated and extensive literature in this review, concerning new findings from tropical plants, including the characterization of endophytic fungi and bacteria microbiota from several Amazon trees, citrus and medicinal plants among others.

Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

ABSTRACT Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a β-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.

Purification and properties of pi-repressible acid phosphatases from Aspergillus nidulans

Two forms of the pacA-encoded acid phosphatase (designated acid phosphatases I and II) secreted by the mold Aspergillus nidulans grown in low-Pi medium at 37 degrees, pH5.0, were purified to apparent homogeneity by PAGE. The M(r) of the purified enzyme forms were ca 115000 (60000) and 113000 (62000) respectively for forms I and II secreted by strain biA1 and ca 118000 (60000) and 121000 (61000) respectively for forms I and II secreted by strain biA1 pacA1, as determined by exclusion chromatography (number between brackets are the M(r) as determined by SDS-PAGE). All of these purified enzyme forms showed an apparent optimum pH ranging from 6.0 to 6.5 and no deviation from Michaelis kinetics for the hydrolysis of both p-nitrophenylphosphate and alpha-naphthylphosphate. Heat inactivation at 60 degrees and at pH6.0 showed half-lives of 14min (k=0.033min(-1)) and 10min (k=0.069min(-1)), respectively, for the purified acid phosphatases I and II secreted by biA1 strain and half-lives of 0.8min (k=0.92min(-1)) and 0.6min (k=0.95min(-1)), respectively, for the purified forms I and II secreted by the biA1 pacA1 strain. The neutral sugar content of purified acid phosphatases I and II secreted by strain biA1 was 48% and 37% (w/w), respectively, whereas the content of forms I and II secreted by strain biA1 pacA1 was 18% and 11%, respectively.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re‐isolated in populations ranging from 106 to 109 CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans

In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae.

Ambient pH-regulated enzime secretion in endophytic and pathogenic isolates of the fungal genus Colletotrichum

In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit.

High molecular diversity of the fungus Guignardia citricarpa and Guignardia mangiferae and new primers for the diagnosis of the citrus black spot

RAPD markers were used to investigate the distribution of genetic variability among a group of Guignardia citricarpa, G. mangiferae, and Phyllosticta spinarum isolates obtained from several hosts in Brazil, Argentina, Mexico, Costa Rica, Thailand, Japan, United States and South Africa. Pathogenic isolates G. citricarpa Kiely (anamorph form P. citricarpa McAlp Van Der Aa) are the etiological agent of the Citrus Black Spot (CBS), a disease that affects several citric plants and causes substantial injuries to the appearance of their fruits, thus preventing their export. Several previous studies have demonstrated the existence of an endophytic species with high morphological similarity to the causal agent of CBS that could remain latent in the same hosts. Consequently, the identification of the plants and fruits free from the causal agent of the disease is severely hampered. The RAPD analysis showed a clear discrimination among the pathogenic isolates of G. citricarpa and endophytic isolates (G. mangiferae and P. spinarum). In addition, a Principal Coordinate Analysis (PCO) based on a matrix of genetic similarity estimated by the RAPD markers showed four clusters, irrespective of their host or geographical origin. An Analysis of Molecular Variance (AMOVA) indicated that 62.8% of the genetic variation was found between the populations (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Substantial variation was found in the populations (37.2%). Exclusive RAPD markers of isolates of G. citricarpa were cloned, sequenced and used to obtain SCARS (Sequence Characterized Amplified Regions), which allowed the development of new specific primers for the identification of G. citricarpa PCR (Polymerase Chain Reaction) analysis using a pair of primers specific to pathogenic isolates corroborating the groupings obtained by the RAPD markers, underscoring its efficiency in the identification of the causal agent of CBS.

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30 degrees C. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the K(m) and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the K(m) and Hill coefficient values were 0.44 mM and 0.97, respectively), beta-glycerol phosphate (the K(m) and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the K(m) and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg(2+), Zn(2+) and Tris-HCl buffer, and is inhibited by Be(2+), histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70 degrees C (half-life of 19.0 min, k = 0.036 min(-1)) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min(-1)) in the same experiment.