Low Inoculum Concentrations Research Articles

Bacterial cellulose from kombucha: Assessing inoculum age and concentration, and its conversion via enzymatic hydrolysis into cellobiose and glucose

Kombucha-type fermentation yields significant amounts of bacterial cellulose alongside the fermented beverage. This study evaluated the effects of inoculum age and concentration in kombucha-type fermentation using green acerola residue as an extract. The findings revealed that lower inoculum concentrations (2.5 and 5%) resulted in decreased bacterial cellulose (BC) yield. Conversely, increasing the inoculum concentration to 7.5% and 10% led to a 3.2-fold increase in BC yield. Inoculums aged just 3 days exhibited the lowest BC production, followed by older inoculums (9 and 12 days), suggesting reduced metabolic activity in the latter case. The optimal combination was found to be an inoculum age of 6 days and a concentration of 7.5%, resulting in a BC yield of up to 2.2 g/L using 70 g/L of carbon source. BC obtained from fermentation underwent enzymatic hydrolysis to yield cellobiose. Multi-stage hydrolysis - 1 stage (48 h), 2 (24 h, 24 h), 3 (12 h, 24 h, 24 h), and 4 (6 h, 6 h, 12 h, 24 h) - utilizing a methodology that anticipates the adsorption of endoglucanases onto the BC itself was employed to mitigate the inhibition of cellobiose on endoglucanases. The stages demonstrated an increase in cellobiose yield corresponding to the greater number of cycles, with 4 stages yielding 12.4% compared to 8.6% for hydrolysis conducted in a single stage. Despite the observed increase, the yield can be considered low and attributed to the loss of adsorbed enzymes between cycles due to the high crystallinity of the BC.

Response of the mushroom pathogen Cladobotryum mycophilum to the fungicides prochloraz and metrafenone and two Bacillus-based biological control agents in mushroom crop trials

Cobweb disease caused by members of the Cladobotryum genus is a major problem for growers of the white button mushroom (Agaricus bisporus). Synthetic fungicides such as prochloraz and metrafenone have been very successful at targeting and eliminating the pathogens that cause mushroom disease. However, prochloraz can no longer be used in the European Union (EU) from June 2023 and over-reliance on metrafenone has resulted in putative resistant pathogenic strains emerging. Prochloraz still showed good control of two different isolates of Cladobotryum mycophilum with efficacy values consistently reaching 70%. Metrafenone inhibited the growth of C. mycophilum isolate 618, which was isolated before metrafenone was introduced (efficacy 96%), but it failed to control C. mycophilum 1546, which was isolated after metrafenone was introduced, and which should now be classified as resistant. Two further C. mycophilum isolates from mushroom farms in 2019 also showed metrafenone resistance in vitro. In this work two biological control agents (BCAs) were investigated as potential environmentally sustainable alternatives to the fungicides prochloraz and metrafenone. The BCA Bacillus velezensis QST 713 was unsuccessful in controlling cobweb disease caused by C. mycophilum isolate 1546 while the BCA Bacillus velezensis Kos showed moderate control over two trials reaching 30–40% efficacy. Lower inoculum concentrations resulted in slightly lower but not significantly different disease levels across all treatments. Future trials with BCAs need to look at alternative methods to evaluate efficacy.

Development and application of a droplet digital PCR assay for the detection of Fusarium oxysporum f. sp. cubense tropical race 4 from different biological samples

AbstractFusarium wilt of banana, caused by the pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a threat to food security as it affects the global banana industry. Control of this pathogen is complex due to the lack of resistant banana or plantain genotypes, lack of effective commercial fungicides, the production of chlamydospores, late appearance of symptoms in infected plants, high adaptability of the pathogen to diverse soil conditions and the polycyclic nature of the disease. Currently, the management strategy for Foc TR4 is exclusion and eradication, which is costly for the producer and the environment because the plants are treated with glyphosate and the affected area is quarantined for a long time. Thus, the development of early detection strategies for Foc TR4 from environmental samples and asymptomatic tissues with low inoculum concentration is essential; this would enable restriction of pathogen spread and minimize the environmental impact by eradication only in areas with accurate data of the pathogen's presence. In this study, three different PCR technologies (conventional PCR, quantitative PCR and droplet digital [dd] PCR) were evaluated for Foc TR4 detection in complex environmental samples. We report the development of a sensitive and specific ddPCR primer/probe set and protocols that can be used as a tool for Foc TR4 detection from symptomatic and asymptomatic plant tissue without prior DNA extraction. The limits of detection in drainage waters, footbaths and soil samples were evaluated using artificial inoculation assays. Finally, environmental samples from Foc TR4‐affected fields were analysed.

Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealedthat larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Development of optimized Verticillium longisporum inoculation techniques for canola (Brassica napus)

Verticillium stripe, caused by Verticillium longisporum, is an emerging soilborne disease of canola (Brassica napus) in Canada. Improved inoculation protocols will facilitate study of Verticillium stripe and its management. Two inoculation techniques, a root-dip method at two inoculum concentrations and a grain inoculation method at four inoculum concentrations, were compared under greenhouse conditions with the canola cultivars ‘45H31’, ‘CS2000’ and ‘Westar’. Symptoms of Verticillium stripe appeared at early growth stages following root dip inoculation, resulting in seedling mortalities of up to 19.7%, 39.5% and 33.3% for ‘CS2000’, ‘45H31’ and ‘Westar’, respectively, at 35 days post-inoculation. Plants inoculated by the root-dip method incurred much greater mortality at the high versus low inoculum concentration. In the surviving adult plants, ‘45H31’ was moderately resistant while ‘CS2000’ was more susceptible to V. longisporum. The grain inoculation method did not cause early-stage mortality, although Verticillium stripe severity at the adult stage was significantly different between control and high inoculum concentrations for all cultivars. In addition, plant dry weight and height decreased as inoculum concentration increased. Inoculation of 1-week-old, 2-week-old and 3-week-old seedlings of ‘CS2000’ and ‘Westar’ was also compared by the root-dip method. The impact of host age at inoculation on disease development was not significant, although higher disease severity was observed at later inoculation in ‘CS2000’. The application of grain inoculum may be more suitable for large-scale screening or studies conducted under field conditions.

High-throughput and point-of-care detection of wheat fungal diseases: Potentialities of molecular and phenomics techniques toward in-field applicability

The wheat crop is one of the most cultivated and consumed commodities all over the world. Fungal diseases are of particular concern for wheat cultivation since they cause great losses and reduced quality, and also for the accumulation of toxin compounds into the final product. In this scenario, optimal disease management strategies are a key point to boosting food production and sustainability in agriculture. Innovative and point-of-care diagnostic technologies represent a powerful weapon for early detection of fungal pathogens and preventively counteract diseases on wheat with the aim to drastically reduce the fungicides as inputs. Indeed, in-field diagnostics devices are fast, sensitive, and ready-to-use technologies able to promptly detect a low inoculum concentration even at the pre-symptomatic stage of the disease. Promising isothermal molecular and phenomics-based methods have been developed to detect wheat fungal pathogens directly in the field. Such technologies could be potentially coupled to directly detect the presence of a certain pathogen and indirectly disclose the plant-pathogen interactions since spectral-based methodologies detect host perturbations following the infection. The present review reports the main in-field isothermal molecular-based and phenomics-based detection technologies for fungal pathogens in wheat discussing their advantages, disadvantages, and potential applications in the near future.

A Methodology for Investigating Aerosolization of Nontuberculous Mycobacteria From Contaminated Heater Cooler Devices

Aerosols inadvertently generated by the bubbling of contaminated water in heater cooler devices (HCDs) have been associated with hundreds of patient infections. These aerosols are typically generated by bubbling within the water tanks of HCDs that subsequently escape from the device leading to contamination of the operating room and potentially infect patients undergoing cardiothoracic surgery. Although these infections are now well-reported in literature, very little research has been done to understand the influence of bubbling frequencies, differences in aerosolization across various species of bacteria, sampling methodologies, infectious dose, etc. Here we report on the development of a miniaturized bubbler with a footprint much smaller than HCDs, that will enable academic researchers and HCD developers to investigate these parameters. Using this bubbler, we found that the aerosolization potential of slow growing Mycobacterium chimaera is about one hundred-fold more (p < 0.05) compared to rapid growing M. smegmatis for low inoculum concentrations (≤106 CFU/mL), underscoring the need for using clinically relevant M. chimaera for evaluating the aerosolization potential of nontuberculous mycobacteria (NTM) from HCDs.

Development of an intelligent laser biospeckle system for early detection and classification of soybean seeds infected with seed-borne fungal pathogen (Colletotrichum truncatum)

There is a need for developing rapid and non-destructive techniques for the early detection of seed-borne fungal pathogen because they can be an essential step towards adopting effective disease control measures. Existing techniques for detecting seed-borne diseases have poor sensitivity towards early stages of pathogen development (i.e., when seeds are asymptomatic) and they are also expensive, time-consuming, complex, require mycological skills and destructive testing operations. Aiming at overcoming the above limitations of the existing techniques, a novel laser biospeckle based method is proposed for early detection of seed-borne fungal infection in conjunction with machine learning. Soybean seeds infected by low concentrations (102-106 spores ml−1) of Colletotrichum truncatum were analysed by using full field biospeckle analysis to establish the possible relationship between biological activity in early stages of pathogen infection, with and without the use of frequency filtering. The results demonstrate that the biospeckle activity (BA), for both, raw and frequency filtered data was significantly high (p < 0.05) for the diseased seeds even for low inoculum concentrations. Moreover, the amplitude values of mid frequency spectral components for diseased seeds were higher than those of lower and higher spectral components which correspond to the BA of fungal infected seeds. Several classical machine learning algorithms were trained to model the response of healthy and diseased samples after parameter optimisation. Obtained results showed that k-nearest neighbour (k-NN), decision tree (DT), and artificial neural network (ANN) based predictive models presented strong robustness and high performance with overall accuracy reaching up to 96.94% for classifying diseased seeds.

The tripartite consortium of Serendipita indica, Trichoderma simmonsii, and bell pepper (Capsicum annum)

Bell pepper (Capsicum annuum) is one of the most widely grown vegetables worldwide. Human health and environmental issues have raised the tendency to use biofertilizers⁄biopesticides as valuable tools in sustainable agriculture. The application of bioinoculants with the potential of plant growth promotion and biocontrol activity against diseases should be a promising alternative for the future of production systems. A combination of two compatible micro-organisms, Serendipita indica and Trichoderma simmonsii, were evaluated for their plant growth promotion/and biocontrol activity against Phytopthtora capsici on bell pepper. We also examined the impacts of varying inoculum concentrations of S. indica on the vegetative growth and against the pathogen at different phenologic stages of the plant. While we recorded a constant range of “percent root colonization” (55–56%) in response to varying inoculum concentrations across all the treatments, light microscopy observation showed the significant effect of the inoculum levels on the root colonization density by the fungus. Seed germination and germination earliness were not affected by individual or the consortium of the bioinoculants, however, a significant negative effect on seed germination was recorded with the increase of inoculum concentrations. Seed inoculation with lower inoculum concentrations (1–3% w/w) of S. indica brought about a significant increase in the vegetative growth of the plant in the greenhouse and field conditions. However, greater concentrations either did not significantly stimulated the plant growth or had less positive effects in comparison to lower ones. Greenhouse and field trials showed S. indica to have a greater impact on the growth parameters in comparison to T. simmonsii or combined use of the bioinoculants. Both the consortium of the fungi and S. indica brought about a significant increase of up to 67% in the bell pepper yield. In the case of bioprotection of the plant against P. capsici pre-emergence seed rot and plantlet blights, the most effective was the consortium of the bioinoculants in terms of declining the disease index. Therefore, we suggest this combination can provide a promising feature as a complement for crop nutrition and enhancing the plant tolerance to P. capsici.

Targeting fish spoilers Pseudomonas and Shewanella with oregano and nettle extracts

To control Pseudomonas and Shewanella as important psychrotrophic spoilage bacteria in fish meat, we used ethanolic extracts of oregano (Origanum vulgare subsp. vulgare) and nettle (Urtica dioica), with phytochemical characterisation of the extracts and their bioactive compounds. Liquid chromatography coupled with photodiode array detection and electrospray ionisation–mass spectrometry was used for qualitative compositional determination of the extracts. Four main compounds were identified in the oregano extract, with rosmarinic acid the most abundant, followed by three glycosylated phenolics, one of which is reported for the first time in O. vulgare: 4′-O-β-d-glucopyranosyl-3′,4′-dihydroxybenzyl-4-hydroxybenzoate. Six main compounds were identified in the nettle extract, as caffeoylmalic acid and five flavonoid glycosides. These oregano and nettle ethanolic extracts showed in-vitro antimicrobial activities against selected Pseudomonas and Shewanella strains in broth and fish meat homogenate when evaluated at two inoculum concentrations. The antimicrobial activities were more pronounced for the nettle extract at the lower inoculum concentration, and for both the Shewanella strains. Growth inhibition in the fish meat homogenate was evaluated at 3.13 mg/mL and 1.56 mg/mL at 5 °C. Again, the nettle extract showed greater antimicrobial activity, which was seen as the lowest maximum growth rate, followed by the oregano extract, which was inhibitory only at 3.13 mg/mL. Finally, the extracts were applied to fish meat that was then stored at 5 °C for 9 days. Evaluation here was for the counts of the mesophilic, psychrotrophic, Pseudomonas and H2S producers. These confirmed the better antimicrobial effects of the nettle extract, especially against the H2S-producing bacteria, which included Shewanella. Both of the extracts were rich in glycosides of flavonoids and phenolic acids. The enzymatic activities of the Pseudomonas and Shewanella spoilage bacteria and their actions on the phenolic glycosides from natural sources will be further investigated.

Effect of inoculum concentration on methanogenesis by direct interspecies electron transfer: Performance and microbial community composition

To understand the effect of inoculum concentration on direct interspecies electron transfer (DIET) for methanogenesis, batch-type anaerobic bioreactors with different inoculum concentrations were operated with and without supplemented granular activated carbon (GAC). With decrease in inoculum concentration, GAC-supplemented bioreactors showed faster methane production rates and reduced lag times. Geobacter species were specifically enriched on the GAC surfaces under lower inoculum concentration conditions. Together, the relative abundance of aceticlastic methanogens (competitors of Geobacter species for acetate) gradually decreased when the inoculum concentration increased. These results suggested that the specific enrichment of Geobacter species by outcompeting with aceticlastic methanogens through low inoculum concentrations on GAC surfaces accelerated methanogenesis by DIET via GAC in anaerobic bioreactors. Taken together, the results of this study suggested that inoculum concentration is an important factor in stimulating DIET for methane production.

Development of Plasmodiophora brassicae in the root cortex of cabbage over time

Pathogen development in the root cortex of three cabbage (Brassica oleracea var. capitata) cultivars that differed in clubroot reaction (susceptible ‘Bronco’, partially resistant ‘B-2819’, resistant ‘Tekila’) was assessed over time in plants inoculated with 1 × 106, 1 × 107 and 2 × 108 spores mL−1 of Plasmodiophora brassica in a controlled environment study. Cortical colonization and symptom development in ‘Bronco’ and ‘B-2819’ were affected by inoculum concentration. When inoculated at 1 × 106 and 1 × 107 spores·mL−1, clubroot levels were generally higher on ‘Bronco’ relative to ‘B-2819’. However, there were no differences at 2 × 108 spores because incidence and severity in both ‘Bronco’ and ‘B-2819’ were near the maximum. Similarly, the area colonized by P. brassicae (%) on root cross-sections was much higher in ‘Bronco’ than ‘B-2819’ at 1 × 106 spores, but differences in cortical colonization, if any, were small at higher inoculum concentrations. The concentration of P. brassicae DNA in inoculated roots (assessed using qPCR) was higher in ‘Bronco’ relative to ‘B-2819’ at 1 × 106 spores, and there were no differences between the two cultivars at the highest inoculum concentration, which matched with the pattern of symptom development. In contrast, symptom development in ‘B-2819’ lagged behind increases in DNA level at 1 × 107 spores. No cortical infection was observed in ‘Tekila’ at the two lower inoculum concentrations, and only a few spindle clubs developed at the highest concentration. These results demonstrated that this source of partial resistance was strongly affected by inoculum concentration, while strong resistance was much less affected.

The Distribution of Listeria in Pasture-Raised Broiler Farm Soils Is Potentially Related to University of Vermont Medium Enrichment Bias toward Listeria innocua over Listeria monocytogenes.

The occurrence of Listeria monocytogenes has been widely investigated in the poultry production chain from the processing plant to the final product. However, limited data are available on Listeria species, including Listeria monocytogenes, in the poultry farm environment. Therefore, fecal and soil samples from 37 pastured poultry flocks from 10 all-natural farms over 3 years were assessed to determine the prevalence and diversity of Listeria within these alternative poultry farm environments using standard cultural and molecular methods. Listeria species were isolated in 15% of poultry farm samples and included Listeria innocua (65.7%), L. monocytogenes (17.4%), and Listeria welshimeri (15.1%). Additional multiplex PCR serotyping showed group 1/2a-3a to be the most dominant L. monocytogenes serovar group. Based on these results, monoculture growth experiments were conducted on four Listeria soil isolates (three L. monocytogenes isolates representing the three recovered serovar groups and one L. innocua isolate) to determine if culture medium [tripticase soy broth (TSB) and University of Vermont modified Listeria enrichment broth (UVM)], inoculum concentration (102 or 105 CFU/ml), or incubation temperature (20, 30, and 42°C) differentially affected these Listeria species. Overall, very few significant growth differences were observed between the behavior of the three L. monocytogenes isolates (representing the three recovered serovar groups) under the growth conditions tested. Alternatively, at 30°C in UVM with the lower inoculum concentration, the L. innocua isolate had a significantly shorter lag phase than the L. monocytogenes isolates. In coculture growth studies under these same incubation conditions, the lag phase of L. innocua and L. monocytogenes was similar, but the final concentration of L. innocua was significantly higher than L. monocytogenes. However, cocultures in UVM for high inoculum concentration did not show preferential growth of L. innocua over L. monocytogenes. These results indicate that the use of UVM as an enrichment medium may preferentially allow L. innocua to outcompete L. monocytogenes at low concentrations, biasing the Listeria prevalence from these farm samples toward L. innocua and potentially underreporting the presence of L. monocytogenes in these environments.

Environmentally Relevant Inoculum Concentrations Improve the Reliability of Persistent Assessments in Biodegradation Screening Tests.

Standard OECD biodegradation screening tests (BSTs) have not evolved at the same rate as regulatory concerns, which now place an increased emphasis on environmental persistence. Consequently, many chemicals are falsely assigned as being potentially persistent based on results from BSTs. The present study increased test duration and increased inoculum concentrations to more environmentally relevant levels to assess their impact on biodegradation outcome and intratest replicate variability for chemicals with known environmental persistence. Chemicals were assigned to potential persistence categories based on existing degradation data. These more environmentally relevant BSTs (erBSTs) improved the reliability of persistence assignment by reducing the high variability associated with these tests and the occurrence of failures at low inoculum concentrations due to the exclusion of specific degraders. Environmental fate was determined using a reference set of 14C-labeled compounds with a range of potential environmental persistences, and full mass balance data were collated. The erBST correctly assigned five reference chemicals of known biodegradabilities to their appropriate persistence category in contrast to a standard OECD Ready Biodegradation Test (RBTs, P < 0.05). The erBST was significantly more reproducible than an OECD RBT (ANOVA, P < 0.05), with more consistent rates and extent of biodegradation observed in the erBST.

Effect of growth conditions on advantages of hup− strain for H2 photoproduction by Rubrivivax gelatinosus

H2 photoproduction by growing cultures of hup− mutant and parental strain RL2 of Rubrivivax gelatinosus was compared. We checked the influence of different substrates, presence of air and N2, culture shaking, inoculum concentrations. At low inoculum concentration, hup− strain demonstrated significant advantage over the parental strain in microaerobic conditions, while under N2–Ar atmosphere it was lower and vanished in anaerobic conditions (Ar only). This advantage was evident when using substrates with low degree of reduction (malate and succinate). Culture shaking under microaerobic conditions and in presence of N2 completely prevented H2 production by both strains. The high inoculum concentration inhibited H2 production under microaerobic conditions and in presence of N2, unlike to anaerobic conditions. With inoculum concentration increase, H2 production decreased not gradually but stepwise which means some metabolic shift. H2 production by hup− strain seems to be more tolerant to air traces than by parental strain.

Extract from Maize (Zea mays L.): Antibacterial Activity of DIMBOA and Its Derivatives against Ralstonia solanacearum.

Many cereals accumulate hydroxamic acids involved in defense of plant against various fungi, bacteria, and insects. 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one, commonly known as DIMBOA, is one of the principal cyclic hydroxamic acids in aqueous extracts of maize. The aim of this study was to evaluate the antibacterial activity of the isolated DIMBOA and its derivatives 2-benzoxazolinone (BOA), 6-chloro-2-benzoxazolinone (CDHB), and 2-mercaptobenzothiazole (MBT) against Ralstonia solanacearum. MBT showed the strongest antibacterial activity, followed by CDHB and DIMBOA, with minimum inhibitory concentrations (MICs) of 50, 100 and 200 mg/L, respectively, better than the BOA with 300 mg/L. These compounds also significantly affect bacterial growth, reduce biofilm formation, and inhibit swarming motility within 24 h. This paper is the first to report the anti-R. solanacearum activity of DIMBOA from Z. mays. The bioassay and pot experiment results suggested that DIMBOA and its derivatives exhibit potential as a new matrix structure of designing target bactericide or elicitor for controlling tobacco bacterial wilt. Further studies must evaluate the efficacy of DIMBOA and its derivatives in controlling bacterial wilt under natural field conditions where low inoculum concentrations exist.

Effect of inoculum concentrations of Aspergillus flavus and A. parasiticus on aflatoxin accumulation and kernel infection in resistant and susceptible maize hybrids

Over a 3-years period, we compared aflatoxin accumulation and kernel infection in maize hybrids inoculated with six inoculum concentrations of Aspergillus flavus isolate NRRL 3357 or A. parasiticus isolate NRRL 6111 which is a norsolorinic acid producer. Aflatoxin resistant and susceptible maize hybrids were inoculated with six conidial concentrations (ranging from 1 × 103 to 1 × 108/ml) of either A. flavus or A. parasiticus using the side-needle technique 7 days after midsilk (50 % of the plants had silks emerged). Aspergillus kernel infection and aflatoxin contamination was determined at ca. 63 days after midsilk. For each hybrid, aflatoxin levels were very similar regardless of the inoculum concentration. With the exception of the A. parasiticus treatments in 2007, the lowest inoculum conidial concentration (1 × 103/ml) for both fungal species was sufficient to produce statistical differences between the resistant and the susceptible maize hybrids for aflatoxin contamination. There was more variability in kernel infection between inoculum concentrations than that observed in aflatoxin accumulation. Since our aflatoxin evaluations are conducted in a warm, humid environment, putting less disease pressure on plants via lower inoculum concentrations may make it possible to identify genotypes that have moderate levels of resistance to aflatoxin accumulation.

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In order to use the residue from the beneficiation of Brazil nuts (Bertholletia excelsa HBK) as substrate in solid-sate fermentation (SSF), in this work, the production of cellulase and xylanase by the fungus Trametes sp. was investigated, using the residue as a carbon source. Employing a 24-1 fractional experimental design, the influence of substrate moisture, nutrient addition and inoculum quantity on enzymatic activities was verified. Moisture was detected to be statistically significant for the production of both enzymes, and increasing the moisture leads to the improvement of cellulase and xylanase activities. Nitrogen and phosphate were also important for enzymes production by the Amazon Trametes sp. The use of this Amazon strain to obtain cellulase and xylanase via SSF of Brazil nut residue appears to be feasible when maintaining substrate moisture at 80%, nitrogen source at 0.9% and low inoculum concentrations. Key words: Cellulolytic activity, xylanolytic activity, Trametes sp., experimental design.

The effect of post‐harvest storage conditions on the development of black dot (Colletotrichum coccodes) on potato in crops grown for different durations

The effects of post‐harvest curing and storage temperature on severity of black dot, caused by Colletotrichum coccodes, were investigated for potato crops grown for different crop durations (days from 50% emergence to harvest) in soils that posed a low, medium and high risk of disease. In field trials over four growing seasons (2005–8), black dot severity at harvest increased with increasing crop duration, within the range 103–146 days from 50% emergence to harvest (P &lt; 0.05). In field trials over three growing seasons (2006–8), black dot severity on tubers at harvest increased significantly with increasing soil inoculum in each year, within the range 43–4787 pg C. coccodes DNA/g soil (P &lt; 0.05). Storage trials were conducted to measure the influence of accumulated post‐harvest temperature on black dot. In 2005, no difference in black dot severity was observed on tubers stored for 20 weeks at 2.5 and 3.5 °C. In 2006 (but not 2007), increasing the duration of curing after harvest from 4 to 14 days increased black dot severity on tubers from 8.9 to 11.2% (P &lt; 0.01) in long duration crops (&gt;131 days after 50% emergence) grown under high (&gt;1000 pg C. coccodes DNA/g soil) soil inoculum. The number of days of curing did not affect disease severity for shorter duration crops grown at high soil inoculum, or on crops grown at medium or low (100–1000 and &lt;100 pg C. coccodes DNA/g soil, respectively) soil inoculum concentrations. Soil inoculum and crop duration together provided a reasonable prediction of black dot severity at harvest and after a 20‐week storage period.

Real-Time PCR Assay for Detection of Sphacelotheca reiliana Infection in Maize (Zea mays) Seedlings and Evaluation of Seed Treatment Efficacy.

Head smut of maize, caused by the fungus Sphacelotheca reiliana, is an economically important disease in all major maize-producing countries. Although seed treatments are commonly used for management purposes, evaluating these treatments for efficacy is both time consuming and inefficient. Therefore, in order to improve the capacity for evaluating seed treatment fungicides, we developed a real-time PCR-based seedling assay for S. reiliana infection. We optimized growth chamber conditions and inoculation methods to achieve infection incidence of 60 to 80% in inoculated, nontreated controls. The effects of five commercially available fungicidal seed treatment formulations on seedling infection incidence were compared by PCR analysis of root and mesocotyl tissues. Tebuconazole, fludioxonil, sedaxane, and Maxim Quattro (fludioxonil+mefenoxam+azoxystrobin+thiabendazole) reduced the incidence of infection (P < 0.05) compared with the control, but no difference was found between the azoxystrobin treatment and the control. All rates tested for both sedaxane and tebuconazole were equally effective for seeds coated with 106 teliospores∙seed-1. Sedaxane, at a rate of 0.1 mg/kernel, eliminated seedling infection if seeds were infested with a low inoculum concentration (101 teliospores∙seed-1). The assay developed here is a valuable tool not only for the detection of fungal infection at the seedling stage, but also for testing the relative efficacies of seed treatments for reducing incidence of infection.