Isolates of 85 bacteria and 94 streptomycete and 35 nonstreptomycete actinomycetes were obtained from a lettuce-growing field in Al-Ain, United Arab Emirates, on colloidal chitin agar, and screened for their ability to produce chitinase. Twenty-three bacteria and 38 streptomycete and 15 nonstreptomycete actinomycete isolates produced high levels of chitinase and were examined in vitro for their ability to suppress the growth of Sclerotinia minor, a pathogen causing basal drop disease of lettuce. The three most suppressive isolates were examined further for their production of β-1,3-glucanase and antifungal activity as well as their ability to colonize the roots and rhizosphere of lettuce in vitro and in planta. The three isolates, Serratia marcescens, Streptomyces viridodiasticus and Micromonospora carbonacea, significantly reduced the growth of S. minor in vitro, and produced high levels of chitinase and β-1,3-glucanase. Streptomyces viridodiasticus also produced antifungal metabolite(s) that significantly reduced the growth of the pathogen in vitro. When the pathogen was presented as the sole carbon source, all three isolates caused extensive hyphal plasmolysis and cell wall lysis. Serratia marcescens and St. viridodiasticus were competent to varying degrees in colonizing the roots of lettuce seedlings after 8 days on agar plates and the rhizosphere within 14 days in pots, with their competency being superior to that of M. carbonacea. All three isolates, individually or in combination, were antagonistic to S. minor and significantly reduced incidence of disease under controlled glasshouse conditions.

Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes

Of 24 Trichoderma isolates, T. harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and β-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa β-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (β-1,3-glucanase). Km and Kcat were 3.8 g l–1 and 0.71 s–1 for the chitinase (chitin) and 1.1 g l–1 and 52 s–1 for the β-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the β-1,3-glucanase was specific for β-1,3-linkages in polysaccharides. Both enzymes were stable at 30°C, while at 60°C the chitinase and the β-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 µg/ml.

Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4).

Structure and function of enzymes acting on chitin and chitosan

In the barley malting process, partial hydrolysis of β-glucans begins with seed germination. However, the endogenous 1,3-1,4-β-glucanases are heat inactivated, and the remaining high molecular weight β-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous β-glucanases to reduce the β-glucans already present in the malt barley is highly desirable. The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of β-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the β-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-β-glucan (barley β-glucan), but showed only residual activity against 1,3-β-glucan (laminarin), or no activity at all against 1,4-β-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-β-glucan were in the ranges of 4–5, and 50–60°C, respectively. The Km and Vmax values for hydrolysis of β-glucan at pH 5.0 and 50°C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower. The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze β-D-glucan that contains both the 1,3- and 1,4-bonds (barley β-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4–5, and temperatures in the range of 50–60°C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.

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Characterization of a β-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry

Despite many achievements in modern agriculture, food crop production continues to be plagued by disease-causing pathogens and pests. In many cases, chemical pesticides effectively protect plants from these pathogens. However, public concerns about harmful effects of chemical pesticides on the environment and human health have prompted a search for safer, environmentally friendly control alternatives (1–3). One promising approach is biological control that uses microorganisms capable of attacking or suppressing pathogens and pests in order to reduce disease injury. Biological control of plant pathogens offers a potential means of overcoming ecological problems induced by pesticides. It is an ecological approach based on the natural interactions of organisms with the use of one or more biological organisms to control the pathogen. Generally, biological control uses specific microorganisms that attack or interfere with specific pathogens and pests. Because of their specificity, different microbial biocontrol agents typically are needed to control different pathogens and pests, or the same ones in different environments. Agriculture benefits, and is dependent on, the resident communities of microorganisms for naturally occurring biological control, but additional benefits can be achieved by introducing specific ones when and where they are needed (4–9). Many agrochemical and biotechnological companies throughout the world are increasing their interest and investment in the biological control of plant diseases and pests. For plant pathogens alone, the current list of microbial antagonists available for use in commercial disease biocontrol includes around 40 preparations (9–11). These are all based on the practical application of seven species of bacteria (Agrobacterium radiobacter, Bacillus subtilis, Burkholderia cepacia, Pseudomonas fluorescens, Pseudomonas syringae, Streptomyces griseoviridis, Streptomyces lydicus) and more than 10 species of fungi (Ampelomyces quisqualis, Candida oleophila,Coniothyrium minitans, Fusarium oxysporum,Gliocladium virens, Phlebia gigantea, Pythium oligandrum, Trichoderma harzianum, and other Trichoderma species). The current market for biological agents is estimated at only $500 million, which is about 1% of the world’s total output for crop protection. The largest share of this market involves biopesticides marketed for insect control (mainly products based on Bacillus thuringiensis

Microbial enzymes in the biocontrol of plant pathogens and pests.

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls.

Ultrastructural and cytochemical studies were carried out on nuclear changes and acrosome formation during the spermiogenesis of the phytophagous bug Euchistus heros . The development of the nucleus involves changes in the shape and in degree of chromatin condensation: initially it is dispersed and with a low-electron density, then assumes a fibrillar arrangement and finally compacts in an electron-dense material. The acrosome is formed by the Golgi complex and presents unusual morphological features during its development. The reaction product of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase activities were detected during various stagesof acrosome development. In contrast, residues of α-N-acetylgalactosamine and basic proteinswere only reported in the intermediate and late stages of the differentiation process, respectively.

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Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae).

Spermiogenesis in the bufonid Melanophryniscus cambaraensis was analysed ultrastructurally. The nucleus of spermatids undergoes morphological modifications divided into five distinct stages based on chromatin condensation. The acrosomal vesicle differentiates into an acrosome and a subacrosomal cone, which cap the anterior region of the nucleus. The spermatozoa consist of the head with a nucleus and acrosomal complex and the tail, with an axoneme, an axial rod and an undulating membrane. The participation of several carbohydrate moieties during this differentiation process was detected using various gold-labelled lectins.

Spermiogenesis in Melanophryniscus cambaraensis (Amphibia, Anura, Bufonidae): ultrastructural and cytochemical studies of carbohydrates using lectins.

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.

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Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae).

The participation of the Golgi complex—endoplasmic reticulum system during spermiogenesis in 3 species of phytophagous bugs (Acrosternum aseadum, Euchistus heros and Nezara viridula) were analysed at the ultrastructural level, after postfixation with osmium tetroxide\potassium iodide. Intense staining was found in the nuclear envelope, endoplasmic reticulum cisternae, Golgi complex, and in a meshwork of tubules scattered throughout the cytoplasm. It is shown that these compartments present a reducing environment. The presence of this kind of staining on the spermatid suggests an important participation of these structures on the differentiation process of this cell.

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A.P. Fernandes

Papers

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls.

Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae).

Spermiogenesis in Melanophryniscus cambaraensis (Amphibia, Anura, Bufonidae): ultrastructural and cytochemical studies of carbohydrates using lectins.

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae).

Contribution of the golgi complex-endoplasmic reticulum system during spermiogenesis in three species of phytophagous bugs (Hemiptera: Pentatomidae)