Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster^™ DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4±3.0×10⁶ and 5.4±3.0×10² CFU ml⁻¹ cell suspension. The detection limit was about 540 CFU ml⁻¹, which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4±3.0×10⁴ CFU g⁻¹ dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.     

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10⁵ to 10¹⁰ CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10⁵ CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB)

A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene ofP. larvae with a micro-scale chip-based real-time PCR system, GenSpector^® TMC-1000, which has uncommonly fast heating and cooling rates (10 °C per second) and small reaction volume (6 μl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54 s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools.     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.     

Real-time PCR method to detect Enterococcus faecalis in water

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.     

Detection of<Emphasis Type="Italic"> Bacillus cereus</Emphasis> group bacteria from cardboard and paper with real-time PCR

The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values (r=0.93, P <0.001) in the range 100–10,000 cfu g^–1. Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10²–10³ bacteria g^–1; and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

Detection of Malaysian methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) clinical isolates using simplex and duplex real-time PCR

The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T _m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.     

Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR

The effect of refrigerated and frozen storage on the viability of Vibrio vulnificus was evaluated using cell suspensions (1 × 10⁸ CFU/ml). Ethidium bromide monoazide (EMA) was utilized to selectively allow real-time (Rti) PCR amplification of target DNA from viable but not dead cells. Bacterial survivors from the EMA Rti-PCR were evaluated by comparison with the plate count assay following different temperature exposures (− 20 and 4 °C) every 24 h for 72 h. The log CFU values from the EMA Rti-PCR assays were erroneously higher than that from plate counts. DNA amplification was not completely suppressed by EMA treatment of low temperature destroyed cells suggesting that membrane damage was not sufficient to allow effective EMA penetration into the cells. The optimal concentration of sodium deoxycholate (SD) was also determined to enhance discrimination of viable and dead cells following exposure of cells to low temperatures. The use of 0.01% or less of SD did not inhibit the Rti-PCR amplification derived from viable bacterial cells. A rapid decrease of the log CFU was observed with cell suspensions subjected to frozen storage and a slow decline in the log CFU occurred at 4 °C. The combination of SD and EMA treatments applied to cells of V. vulnificus held at − 20 °C and 4 °C resulted in a high level of correlation between the log of CFU (plate counts) and the log of the number of viable cells determined from SD+EMA Rti-PCR.     

Characterisation ofEscherichia coli isolated from raw milk cheeses

The aim of this work was: (i) to verify the level ofEscherichia coli in Pannerone and Valtrompia Formaggella, two artisanal Italian raw-milk cheeses ripened for less than 60 days; (ii) to phenotypically and genotipycally type theE. coli isolates; (iii) to detect the presence ofE. coli O157:H7 and of intestinal enteropathogenicE. coli by PCR. The levels ofE. coli in the cheeses ranged from 3.89 to 8.47 log CFU g^?1. NoE. coli O157:H7 was detected in 25 g of cheese. The 76E. coli strains (68 cheese isolates and 8 reference strains) were widely diverse, since a high number of both PCR fingerprinting profiles and PhenePlate® phenotypes were shown. Within the 68 cheese isolates, no toxin production and virulence-associated genes were shown by multiplex PCR. Non-pathogenicE. coli were isolated at high levels in raw-milk cheeses, where they may contribute to the development of desirable characteristics of some of these products, e.g. Pannerone.     

Rapid Real-Time PCR Assay for Culture and Tissue Identification of <Emphasis Type="Italic">Geomyces destructans</Emphasis>: the Etiologic Agent of Bat Geomycosis (White Nose Syndrome)

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS–PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10⁻¹–5 × 10⁷), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)–PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.     

Predictive formulae for goat cheese yield based on milk composition

Prediction of the yield and quality of different types of cheeses that could be produced from a given type and/or amount of goat milk is of great economic benefit to goat milk producers and goat cheese manufacturers. Bulk tank goat milk was used for manufacturing hard, semi-hard and soft cheeses (N = 25, 25 and 24, respectively) to develop predictive formulae of cheese yield based on milk composition. Fat, total solids, total protein and casein contents in milk and moisture-adjusted cheese yield were determined to establish relationships between milk composition and cheese yield. Soft, semi-hard and hard cheeses in this study had moisture contents of 66, 46 and 38%, respectively, which could be used as reference standards. In soft cheese, individual components of goat milk or a combination of two or three components predicted cheese yield with a reasonably high correlation coefficient (R² = 0.73–0.81). However, correlation coefficients of predictions were lower for both semi-hard and hard cheeses. Overall, total solids of goat milk was the strongest indicator of yield in all three types of cheeses, followed by fat and total protein, while casein was not a good predictor for both semi-hard and hard cheeses. When compared with moisture-adjusted cheese yield, there was no difference (P > 0.05) in predicting yield of semi-hard and hard goat milk cheeses between the developed yield formulae in this study and a standard formula (the Van Slyke formula) commonly used for cow cheese. Future research will include further validation of the yield predictive formulae for hard and semi-hard cheeses of goat milk using larger data sets over several lactations, because of variation in relationships between milk components due to breed, stage of lactation, season, feeding regime, somatic cell count and differences in casein variants.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

Microbiological quality and related factors of sheep milk produced in farms of NE Greece

Sheep herds are a significant capital of dairy industry in Greece, but as dairy farming becomes more complex and intense the need to provide assistance to dairy producers on milk quality is critical. In this study, the quality of raw milk produced in sheep farms of NE Greece was surveyed and the role of various factors was explored. In total, 21 dairy ewe’s farms from the regions of Xanthi and Evros, in the north-eastern Greece were monitored. Milk samples were sampled after the morning milking every 15 days throughout the dairy period (March–June). For the study, a questionnaire was also filled by personal interview with the owners in order to collect information about herd characteristics, health status, handling practices etc. From each farm, air was also sampled for microbiological analysis. Milk samples were examined for chemical components: fat content, protein, lactose, non-fat dry matter (NFDM) and somatic cells count (SCC). Microbiological examination involved the estimation of Total Bacterial Counts (TBC), coliform count (CC), Staphylococcus aureus, Streptococcus sp., and preliminary incubation count (PIC). The possible correlation among different bacterial species and their interaction with SCC and chemical components of milk was also considered. It was examined whether farm management practices could influence the hygiene and the quality of milk. Our results show that as an average TBCs were 5.48 log cfu/ml, SCC: 6.05 log cells/ml, CC: 4.49 log cfu/ml, S. aureus: 3.94 log cfu/ml, Streptococcus sp.: 4.95 log cfu/ml and PIC: 5.7 log cfu/ml. The mean fat, protein, lactose and NFDM were 6.17%, 5.28%, 4.73% and 10.95% respectively. The study revealed significant positive correlation between TBC and PIC (0.825), while SCC was marginally positive correlated with protein and NFDM. No statistically significant correlations observed among SCC with any of the bacterial species. Herd size and farm management practices had considerable influence on SCC and bacterial species.     

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in “real time” during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10³ spores and10² spores in talcum powder, respectively, whereas PCR could detect 10⁴ spores in soil and 10³ spores in talcum powder, respectively.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.     

δC, δN, carbon, nitrogen and phosphorus as indicators of plant ecophysiology and organic matter pathways in Everglades deep slough, Florida, USA

Historically, the Florida Everglades was characterized by a corrugated landscape of shorter hydroperiod, elevated sawgrass (Cladium jamaicense) ridges and longer hydroperiod, deep water slough communities. Drainage and compartmentalization of the Everglades have fundamentally altered this pattern, and sawgrass ridge communities have expanded at the expense of deep water slough communities throughout much of the landscape. In this study we provide a simple isotopic and nutrient characterization of major components of the slough ecosystem to elucidate physiological and nutrient differences among species and to suggest pathways for organic matter decomposition that contribute to peat development in deep water sloughs. We examined carbon (C) and nitrogen (N) isotopes and C, N and phosphorus (P) concentrations of the floating-leaved macrophytes Nymphaea odorata and Nymphoides aquatica, the emergent macrophyte Eleocharis elongata, and the submerged species Utricularia foliosa and Utricularia purpurea, as well as soil and flocculent material from the southern Water Conservation Area 3-A. Flocculent material and soils had the highest N content (4.5 ± 0.2%) and U. foliosa and N. odorata had the highest P content (0.13 ± 0.01% to 0.12 ± 0.01%). The range for δ¹⁵N average ± SE values was 5.81 ± 0.29‰ (U. foliosa) to −1.84 ± 0.63‰ (N. odorata), while the range for δ¹³C values was −23.83 ± 0.12‰ (N. odorata) to −29.28 ± 0.34‰ (U. purpurea). Differences of up to 10‰ in C isotopic values of U. foliosa and N. odorata suggest fundamental physiological differences between these species. Along a degradation continuum, enrichment of ¹³C and ¹⁵N and extent of decomposition was negatively related to phosphorus concentrations. A two end-member ¹³C mixing model suggested that Utricularia species were the primary organic source for flocculent materials, whereas organic matter derived from root decomposition of N. odorata contributed to the progressively enriched δ¹³C values found with depth in soils. These results illustrate the fundamentally important roles of Nymphaea and Utricularia species in ecosystem dynamics of deep water sloughs.     

Development of Pusa 5SD for seed dressing and Pusa Biopellet 10G for soil application formulations of Trichoderma harzianum and their evaluation for integrated management of dry root rot of mungbean (Vigna radiata)

Various seed dressing and soil application formulations were developed from Trichoderma viride, T. virens and T. harzianum to increase the shelf life of bio-formulations used to manage dry root rot (Rhizoctonia bataticola) of mungbean (Vigna radiata), a major yield limiting factor in mungbean production. The shelf life of the formulations developed in the present study was monitored by counting colony forming units (cfu) up to 25 months of storage at room temperature (26 ± 8 °C). A newly developed seed dressing formulation, Pusa 5SD based on peat powder (47.5%), Sabudana powder (Manihot esculenta) (47.5%) and carboxymethyl cellulose (5%) and a newly developed soil application formulation, Pusa Biopellet (PBP) based on sodium alginate, aluminium silicate, Sabudana powder and tap water (1:5:5:100 w/w/w/v) exhibited longer shelf life. Another formulation Pusa Biogranule (PBG) based on wheat and pulse brans varied in cfu counts during different periods of storage. Pusa 5SD could be used up to 25 months of storage while PBP 10G and PBG 5 could be used up to 15 months of storage (>10⁵ cfu). The efficacy of the formulations was evaluated in pot experiments against the disease. In these experiments, T. harzianum based PBP 10G and PBG 5 for soil application, and Pusa 5SD for seed treatment were found to be superior to others in reducing the dry root rot incidence, and increasing the seed germination and shoot and root lengths. However, a combination of soil application of PBP 10G (T. harzianum) and seed treatment with T. harzianum based Pusa 5SD + carboxin was found superior to the use of any of these formulations alone in reducing the dry root rot incidence (87.2%) and increasing the seed germination (43.0%), shoot length (40.3%), root length (37.0%) and grain yield (54.6%) of mungbean crop over those of untreated control under sick field conditions.