Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples

A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/μL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 10(5) CFU/g soil(-1) equivalent to 10 pg/μL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.     

Biocontrol potential and polyphasic characterization of novel native <Emphasis Type="Italic">Trichoderma</Emphasis> strains against <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolated from sorghum and common bean

Native strains of Trichoderma isolated from sorghum and common bean crop soils were investigated to assess their biocontrol potential over the phytopathogenic fungus Macrophomina phaseolina, isolated from diseased plants. The Trichoderma strains were characterized with a polyphasic approach, which combined the analysis of their morphological characteristics, enzymatic activity, macro- and microculture test results, rDNA restriction patterns (AFLP), ITS1-5.8S-ITS2 rDNA sequences, and protein profiles. The integration of these data sets can be used to select new isolates as biological control agents against native fungal phytopathogens. In general, we observed a positive correlation between the secretion of beta-1,3-glucanase and N-acetylhexosaminidase, and the biocontrol capacities of all the Trichoderma isolates. Strains with the best hyperparasitic behavior against M. phaseolina isolated from diseased bean and sorghum were Trichoderma sp. (TCBG-2) and Trichoderma koningiopsis (TCBG-8), respectively.     

Variability in antifungal proteins in the grains of maize, sorghum and wheat

Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β-glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β-glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β- and 1,3–1,4-β-glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3-β-glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β-glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β-glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.     

Characterization of an endo-1,3(4)-β-d-glucanase gene from Cellvibrio mixtus

Abstract An endo-1,3(4)-β- d -glucanase gene ( cwd2 ) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb Pst I fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli , degraded both β-1,3 glucans and β-1,3–1,4 mixed-linkage glucans, was entohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40°C respectively. The M _r of specifically labelled Cwd2 was approximately 34 000. This gene was quite distinct from two other C. mixtus β-1,3 glucanases previously described.     

Intercellular proteins and β-1,3-glucanase activity associated with leaf rust resistance in wheat

To investigate biochemical aspects of resistance conferred by the Lr35 gene for adult-plant resistance in wheat ( Triticum aestivum L.) to leaf rust, pathogen development was related to intercellular protein composition and β -1,3-glucanase (EC 3.2.1.39) activities at three growth stages in infected and uninfected resistant (RL6082 [Thatcher/ Lr35 ]) and susceptible (Thatcher) plants. Leaf rust symptoms produced by pathotype UVPrt9 of Puccinia recondita f. sp. tritici showed that resistance conferred by Lr35 was most effective at the flag leaf stage. Furthermore, fluorescence microscopy indicated that resistance was strongly associated with hypersensitive cell death of invaded tissue. According to polypeptide profiles, intercellular proteins with molecular masses of 35, 33, 31 and 26 kDa were constitutively present at higher levels in resistant than in susceptible plants at the flag leaf stage. Four intercellular proteins (35, 33, 32 and 31 kDa) serologically related to β -1,3-glucanase were present in resistant and susceptible genotypes during all stages of plant growth. Resistance was associated with high constitutive levels of β -1,3-glucanase activity. Susceptibility on the other hand was associated with low constitutive levels of β -1,3-glucanase, while high levels were induced by infection during more advanced stages of colonization. Our results suggest that β -1,3-glucanase is involved in the defense response controlled by the Lr35 gene.     

Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina

Pathogenesis-related (PR) proteins are induced in response to pathogen attack. In the present study, the induction of PR proteins in response to the fungal pathogen Macrophomina phaseolina was investigated in 15-day- and 1-month-old plants of Vigna aconitifolia with resistant and susceptible cultivars. Inoculation of the fungal pathogen resulted in the enzyme activity gradually increased throughout the experimental period of 168 h compared to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in the resistant FMM-96 cultivar as compared to susceptible RM0-40 and CZM-3 cultivars. Furthermore, the western blot analysis revealed the presence of 33- and 30-kDa bands of β-1,3-glucanase and chitinase in induced moth bean plants, respectively. The possible implications of these findings as part of the general defense response of moth bean plants against the fungal pathogen (M. phaseolina) have been discussed.     

Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

The role of fungi in the diet of the amphipod Gammarus pulex (L.): an enzymatic study

SUMMARY. 1. Sets of ten Gammarus pulex fed on controlled diets of sterile alder leaves, or fungal mycelium, or alder leaves incubated for 10 days with an aquatic hyphomycete, were assayed for cellulase, β-1,3-glucanase an d chiitinase activity and compared with (a) animals taken directly from the stream, (b) animals starved for 2 days, and (c) enzyme activity in fungal mycelium.
2. Gut enzyme activity was compared on natural substrates of sterile leaves, mycelium and inoculated leaves as well as on model substrates.
3. G. pulex secretes an endogenous coupled cellulase system capable of degrading native cellulose in plant cell walls. It also secretes β-1,3-glucanase and chitinase capable of degrading fungal cell walls thus affording access for gut enzymes to cell contents.
4. Secretion of enzymes active on native cellulose is enhanced on a diet of leaves already partially degraded by fungal enzymes. Gut enzymes extract more reducing sugar from this substrate than from sterile leaves. Specific enzyme secretion is enhanced by the presence in the diet of exposed, accessible substrates. Fungal enzymes do not appear to contribute to the digestive processes of G. pulex.     

Effect of moisture stress, plant population density and pathogen inoculation on charcoal stalk rot of sorghum

The effects of moisture deficit stress, plant population density and pathogen inoculation technique on charcoal stalk rot in the sorghum hybrid CSH 6 were studied in the 1980–81 and 1981–82 post-rainy seasons at three locations in India. Incidence and severity of charcoal rot caused by Macrophomina phaseolina were compared in three plant population densities, subjected to different moisture stress regimes created by withholding irrigation at various plant growth stages. Natural infections were compared to artificial inoculation with M. phaseolina. Combinations of moisture stress, plant population and inoculation treatments were compared to identify the combination most likely to develop maximum disease. Lodging, the first external symptom of charcoal rot, was significantly correlated with other disease symptoms used to measure charcoal rot, such as soft stalk, number of nodes crossed by M. phaseolina infection, root damage and plant senescence. In both seasons the highest incidence of lodging occurred when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage. The greatest incidence of the disease was recorded in the highest plant population (266 700 plant ha^-) at all three locations. No significant differences were found between artificially and naturally inoculated treatments. The maximum number of lodged plants was found at a density of 266 700 plants ha^-1 when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage.     

Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR

Forty isolates of Macrophomina phaseolina, a pathogen causing charcoal dry root rot of soybean, cotton, and chickpea, were genetically characterized with universal rice primers (URP; primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of 12 URPs used in this study, 5 primers were effective in producing polymorphic fingerprint patterns from the DNA of M. phaseolina isolates. Three primers (URP-2F, URP-6R, and URP-30F) were quite informative and produced high levels of polymorphism among the isolates of M. phaseolina. Analysis of the entire fingerprint profiles using unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated M. phaseolina isolates obtained from soybean, cotton, and chickpea hosts into specific groups. In this study, we found for the first time transferability and use of PCR primers derived from plant genomes to generate host-specific fingerprint profiles of M. phaseolina, a broad host range plant pathogenic fungus. These results demonstrate that URPs are sensitive and technically simple to use for assaying genetic variability in M. phaseolina populations.     

Evaluation of Pseudomonas chlororaphis subsp. aurantiaca SR1 for growth promotion of soybean and for control of Macrophomina phaseolina

Pseudomonas chlororaphis subsp. aurantiaca SR1 was evaluated for control of Macrophomina phaseolina in vitro and in soybean plants, for growth promotion of soybean plants and for production of antifungal compounds. Strain SR1 caused a significant inhibition of M. phaseolina in vitro and reduced damping-off in the in vivo assays. In addition, strain SR1 significantly increased shoot and root length and shoot and root dry weight of soybean plants in M. phaseolina infested soil, as compared to control plants in infested soil. Fragments for the phenazine-1-carboxylic acid, pyrrolnitrin and hydrogen cyanide encoding genes were amplified from the DNA of strain SR1 after polymerase chain reaction (PCR) assays with specific primers. Thus, this study establishes that P. chlororaphis subsp. aurantiaca SR1 provides control of M. phaseolina in vivo and suggests that strain SR1 might be applied as an effective biocontrol agent to protect soybean plants from this phytopathogen.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Immunogold localization of beta-1,3-glucanases in two plants infected by vascular wilt fungi.

An antiserum raised against a purified tobacco beta-1,3-glucanase (PR-N) was used to study the subcellular localization of enzyme in fungus-infected plant tissues by means of post-embedding immunogold labeling. In susceptible tomato plants, the enzyme accumulation was found to occur as a result of successful tissue colonization, whereas it appeared to be an early event associated with limited spread of the fungus in resistant tissues. Although marked differences between susceptible and resistant tomato cultivars were observed in the rate of production of beta-1,3-glucanase, the pattern of enzyme distribution was similar. The enzyme was found to accumulate predominantly in host cell walls and secondary thickenings of xylem vessels. By contrast, a very low amount of enzyme was associated with compound middle lamellae. The occurrence of beta-1,3-glucanase at the cell surface of invading fungi was an indication of their possible antifungal activity. A low enzyme concentration was detected in vacuoles of both healthy and infected tissues. In infected eggplant tissue, the pattern of beta-1,3-glucanase distribution was similar to that observed with tomato. Whether these hydrolases accumulate first in vacuoles and are subsequently conveyed toward the outside to participate in fungal wall lysis remains to be determined.     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

Possible role of cAMP in the synthesis of β-glucanases and β-xylanases of Bacillus circulans WL-12

Abstract Bacillus circulans WL-12 secretes 1,4-β- d -xylanase and 1,3-β- d - and 1,6-β- d -glucanase activities. All of them are catabolites regulated by glucose and, while xylanase needs xylan as the inducer, the two latter enzyme activities are formed once glucose is depleted. Cyclic nucleotides such as adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5' monophosphate (cGMP) exhibit a negative effect on enzyme synthesis if added to the culture media. Based on the fact that only cAMP is found in cells growing in glucose-rich media we propose a model for B. circulans WL-12 in which cAMP acts as a negative effector for regulating the synthesis of these enzymes. The model is not, however, extrapolated to other Bacillus species and all B. circulans strains.     

Spatial distribution of chitinases and β-1,3-glucanase transcripts in bean arbuscular mycorrhizal roots under low and high soil phosphate conditions

Arbuscular mycorrhizal (AM) fungi extensively colonize the root cortex under low-soil-phosphate (P) conditions, whereas infection is limited under high-P conditions. Fungal growth under both P conditions might be influenced by plant defence-related gene expression. In this study, we used in situ hybridization methods to compare the cellular localization of three defence-related mRNAs in non-infected bean roots and in relation to fungal infection units. In non-infected and infected roots, mRNAs encoding acidic and basic endochitinases were generally most abundant in the vascular cylinder. High-P-grown mycorrhizal roots showed localized accumulation of the acidic endochitinase mRNA in cortical cells containing arbuscules and in their immediate vicinity (one to five cell layers). The pattern of accumulation of the basic endochitinase mRNA was not affected by P or AM fungal infection. At the low P concentration, the β-1,3-glucanase mRNA accumulated predominantly in the vascular cylinder of non-infected roots. Suppression of β-1,3-glucanase mRNA accumulation in these tissues was observed in non-infected roots at the high-P and in mycorrhizal roots at both P concentrations. The observed suppression extends at least several mm from fungal infection units, characterizing a systemic effect. Beta-1,3-glucanase mRNA accumulated also around a number of cortical cells containing arbuscules only at the low P concentration. The localized accumulations of the endochitinase and β-1,3-glucanase mRNAs suggest that the encoded proteins might be involved in the control of intraradical fungal growth.     

Local and Systemic Induction of β-1,3-Glucanase and Chitinase in Coffee Leaves Protected Against Hemileia vastatrix by Bacillus thuringiensis

β-1,3-glucanase and chitinase activities were induced locally and systemically 4–25 and 11–25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14–18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18–21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemicallyprotected unchallenged coffee leaves.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.