Chemicals for preventing moulding in damp hay

Fewer than one third of more than 100 chemicals tested prevented moulding when 0.5% of active ingredient was added to hay containing 35% water or, alternatively, when 0.02% of active ingredient was added with 0.2% propionic acid or ammonium propionate. Chemicals which prevented moulding in these tests when used alone included aliphatic acids with 2–6 carbon atoms, sorbic acid, short chain aldehydes and phenols, propionic acid, fully or half-neutralized with ammonia, and 8-quinolinol and its derivatives. Formic acid, aliphatic acids with 7–9 carbon atoms, longer chain aldehydes and some fungicides delayed moulding for one to two weeks. Only formaldehyde, 8-quinolinol and its derivatives, and p-nitrophenol increased the activity of 0.2% propionic acid. Although the effective doses of propionic acid, ammonium propionate, and propionic acid, half-neutralised with ammonia, were similar, there was evidence that the presence of free acid in the formulation aided redistribution to overcome undertreatment of pockets in the hay. Further tests of 2-phenyl phenol, phenyl propionate and 8-quinolinol showed that the latter was the best candidate for development. Fungi of the Aspergillus glaucus group were first to colonise most tests that failed, indicating their high degree of tolerance of a wide range of chemicals, and their value as test organisms for prospective preservatives.     

Preservation of baled hay with propionic and formic acids and a proprietary additive

Field-baled hay was incompletely preserved using propionic or formic acids. Results were most satisfactory, although moulding by Aspergillus glaucus group was still frequent, when die hay contained less than 30% water and propionic acid was used. However, much more propionic acid was needed than in laboratory experiments probably because poor distribution of acid in the bale, resulted in under-treated pockets of hay where moulds tolerant to propionic acid could grow and then spread to areas that would otherwise have been adequately treated. In Dewar flasks, moulds, particularly Paecilomyces varioti and the A. glaucus group, could spread from untreated hay into hay containing 2% w/w of propionic acid. Addition of 4 or 10% of propionic acid protected adjacent untreated hay from moulding. The concentration of propionic acid necessary to prevent flasks from heating and moulding increased with thickness of the untreated layers and with water content. Redistribution of acid in the bale after treatment was slow and much variation in acid levels occurred even between adjacent 1 cm segments of grass following storage. Spontaneous heating of moulding hay could cause migration of propionic acid in hay, assisting the spread of fungi. A proprietary additive did not prevent moulding at any hay water content tested.     

Moulding of sugar-cane bagasse and its prevention

When fresh sugar-cane bagasse containing about 50% water and 3% sugar was baled and stacked, it quickly heated to over 50°C and remained hotter than 40°C for 50 days. The residual sucrose was utilized by microbial growth and the content of fungal, bacterial and actinomycete spores increased to more than 10⁸/g dry wt. The spores in heated bagasse were mostly of thermophilic actinomycetes and fungi, and included two actinomycetes implicated in bagassosis. Thermoactinomycetes sacchari occurred in 40% of samples examined, some of which yielded up to 5 × 10⁶ colonies/g, while T. vulgaris occurred in 80% of samples, but these rarely yielded more than 10⁵ colonies/g. Other organisms were cellulolytic and caused fibre deterioration. Heating and moulding could be much decreased either by drying to about 25% water content, which halved the spore content after storage, mostly at the expense of the actinomycetes, or by adding 2% by weight of propionic acid, which decreased the spore content to 4 × 10⁶ spores/g or less even after 18 months' storage. Sometimes adding only 0·6% of propionic acid or 2% of propionic acid applied as a 50% aqueous solution had a similar effect. Treatment with propionic acid thus decreased the deterioration of bagasse, permitted its storage between harvests and prevented the hazard of bagassosis to workers.     

Methods for testing chemical additives to'prevent moulding of hay

Three methods for testing additives for their ability to prevent moulding of hay are described. Storage of 25 g samples of hay in glass jars at 25 ^oC was useful for screening many chemicals rapidly. Dewar flasks (4–5 1) allowed testing on 500 g hay samples with sufficient insulation to allow any effects of chemicals on spontaneous heating as well as on moulding to be measured. Large drums containing 20 kg hay were used for larger scale tests with partially dried fresh, or rewetted old hay. Proprietary additives were unsuccessful at their recommended application rates and some even failed to show antifungal activity with very much larger doses. Volatile fatty acids, particularly propionic acid, and ammonium propionate were the most effective chemicals tested. The amount of propionic acid needed to prevent moulding and spontaneous heating could be defined in terms of the amount of water in the hay. Where the ratio of propionic acid to water was greater than 1–25:100 heating was rare. Ammonium propionate was slightly less effective than propionic acid against moulding. However, it lacks a pungent odour, is less volatile, less corrosive and is more pleasant and safer to handle.     

Evidence that the mechanisms of fungitoxicity of 8-quinolinol and its bischelate with copper(II) are different

The copper(II) contents of the growth media, Sabouraud dextrose and Czapek-Dox broths, and of the spore inocula of Aspergillus niger (ATCC 1004), Aspergillus oryzae (ATCC 1011), Trichoderma viride (ATCC 8678), and Myrothecium verrucaria (ATCC 9095) were determined by atomic absorption spectrophotometry in a graphite furnace. The test systems composed of Sabouraud dextrose broth and spore inocula of the four fungi contained only a little over 3% of the copper(II) required to form a minimal inhibitory concentration of bis(8-quinolinolato)copper(II). The test system of Czapek-Dox broth and A. oryzae contained slightly less than 65% of the copper(II) required to form a minimal inhibitory concentration of the bischelate of 8-quinolinol with copper(II). When the minimal inhibitory concentrations of 8-quinolinol and bis(8-quinolinolato)copper(II) were added simultaneously to the test system of Czapek-Dox broth and A. oryzae, 10% of the combined mixture of toxicants caused complete inhibition of growth indicating synergism between the toxicants. These results together with the observation that alpha-lipoic acid as well as small aliphatic thiol-containing compounds (cysteine, glutathione, dithioerythritol, and dithiothreitol) reversed the toxicity of 8-quinolinol but not the toxicity of bis(8-quinolinolato)copper(II) led to the conclusion that the mechanisms of fungitoxicity of both toxicants are different.     

Survivability of indigenous microflora and a Salmonella typhimurium marker strain in poultry mash treated with buffered propionic acid

Buffered propionic acid (BPA) was evaluated as a potential treatment for the elimination of Salmonella spp. in poultry mash. A primary poultry isolate marker strain of Salmonella typhimurium was added as either a broth or in a dry chalk carrier form to poultry mash containing soybean meal as a protein supplement. The mash was supplemented with buffered propionic acid at 2, 4, 6, 8, 10, 20, 30, 50 and 100 g kg⁻¹ diet and samples were enumerated for indigenous aerobic bacteria, fungi and the S. typhimurium marker strain. Total indigenous aerobic bacteria and fungal populations were generally decreased by addition of more than 20 g BPA kg⁻¹, but an addition of 100 g BPA kg⁻¹ mash was usually required to achieve reductions of approximately 90% of indigenous aerobic bacteria and 99% of indigenous fungi. After 7 days of storage, 8 g BPA kg⁻¹ mash also reduced S. typhimurium populations by more than 90% in mash inoculated via chalk, while at least 50 g BPA kg⁻¹ mash was required to provide the same level of reduction in mash inoculated with a liquid culture of S. typhimurium. Although BPA does not appear to be an overly effective antimicrobial agent with respect to indigenous aerobic bacterial populations in animal feed, higher concentrations may have the potential for reducing fungal and Salmonella spp. contamination in poultry mash.     

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.     

A potent feed preservative candidate produced by Calcarisporium sp., an endophyte residing in stargrass (Cynodon dactylon)

AIMS: The cultures of an endophytic fungus Calcarisporium sp. were screened for inhibitors on the growth of feed-associated moulds and on the aflatoxin biosynthesis to find a safe and effective feed preservative. METHODS AND RESULTS: Eight test fungi were isolated from the spoiled poultry feed. The endophytic fungus Calcarisporium sp. was separated from the Chinese coastal grass Cynodon dactylon. The antifungal action concerning the endophytic culture extract (ECE) was performed with propionic acid (PPA) as the corresponding reference. The ECE had a similar antifungal efficacy to PPA in a concentration-dependent manner. The susceptibility order of the ECE to the test fungi was found to be Fusarium sp. > Aspergillus spp. > Penicillium spp. Furthermore, the application of the ECE in pelleted-layer duck feed as a preservative was carried out at a humidity of 10, 15 and 20%. It has been discerned that mould growth and aflatoxin biosynthesis could be co-inhibited almost completely by ECE at concentrations higher than 1.0% (w/w). The LD50 of the ECE on mice was shown to be higher than 28 g kg-1. CONCLUSIONS: The ECE can be selected as an inhibitor to preserve poultry feed on inhibiting the growth of mould and aflatoxin biosynthesis during feed storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ECE may be an effective and biosafe antifungal ingredient for poultry feed and holds a potential market prospect in feed industry.     

Antifungal Proteins during Sorghum Grain Development and Grain Mould Resistance

Proteins potentially inhibitory to the growth of grain‐moulding fungi in vitro have been identified from sorghum seeds. However, their role in vivo during fungi infection is still not clear. The objective of the present study was to evaluate the presence of antifungal proteins (AFPs) during grain development. Sureño (a grain mould‐resistant line), RTx430 (a grain mould‐susceptible) and their F₁ hybrid, were planted at two moisture levels. Caryopses were collected from each genotype every 7 days after anthesis (DAA) during development and maturity of the grain. Significant levels of grain mould occurred naturally. Levels of four AFPs (sormatin, chitinases, β‐1,3‐glucanases and ribosomal‐inactivating proteins) were determined using the immunoblotting technique. During grain development (7–35 DAA), Sureño and the F₁ hybrid showed higher levels of sormatin and chitinase than RTx430. RIPs levels in Sureño and the F₁ hybrid were higher than those in RTx430 after 21 DAA. Sormatin, chitinases, β‐1,3‐glucanases and RIPs levels in Sureño and in the F₁ hybrid were higher than those of RTx430 after grain physiological maturity. AFPs are associated with grain mould resistance because Sureño and the F1 hybrid induce and/or retain higher AFPs levels under grain mould infection pressure than did RTx430.     

Does endophyte symbiosis resist allelopathic effects of an invasive plant in degraded grassland?

Allelopathic effects and plant associated systemic endophytic fungi are often thought to play a role in the invasion of exotic plant species. Here, we tested the inhibitory effects of the aqueous extracts of the hemiparasitic weed Pedicularis kansuensis on seed germination and seedling growth of endophyte-free (E−) and -infected (E+) grass species, Stipa purpurea and Elymus tangutorum. The weed extracts significantly inhibited both seed germination and seedling growth of the target grass species. Extracts from the inflorescences gave greater inhibition than those from the stems or roots, while the concentration of the extract had a direct effect on the extent of inhibition. The E+ target plants were less susceptible to the extracts than their E-counterparts. Our results suggest that the allelopathic potential of P. kansuensis will lead to increased frequencies of endophyte infected plants in grass populations.     

Protecting wood from mould,decay, and termites with multi-component biocide systems

Biocides must be developed for controlling mould establishment on cellulose-based building materials. Accordingly, biocides intended for indoor applications must be non-toxic, non-volatile, odourless, hypoallergenic, and able to provide long-term protection under conditions of high humidity. Multi-component biocide systems were tested in American Wood-Preservers’ Association soil block tests for inhibition of brown-rot and white-rot decay fungi and American Society for Testing and Materials standard tests for inhibition of mould fungi and termites. Multi-component systems combining a borate base supplemented with either 0.1% azole or 0.5% thujaplicin, performed well against the two brown-rot fungi Postia placenta and Gloeophyllum trabeum; the white-rot fungus Coriolus versicolor; the three mould fungi Aspergillus niger, Penicillium chrysogenum, and Trichoderma viride; and the subterranean termite Reticulitermes flavipes (Kollar). It was concluded that for interior applications borate-based multi-component biocide systems can protect wood from decay fungi, mould fungi, and termites, and that a system containing thiabendazole provided protection at a lower retention than the other biocides in this study. Synergy was observed between the borate base and voriconazole in inhibition of mould.     

Use of fungal metabolites to protect wood-based panels against mould infection

Wood-based composites such as oriented strand board (OSB) are principle framing elements in building construction in North America. However, these materials are often affected by moulds in wet or humid environmental conditions. A common control method for prevention of mould growth on panels is preservative treatment of panels with various pesticides or chemicals. In recent years, environmentally friendly pest control methods are required because of environmental issue. This research aimed to develop a biological technology to protect OSB against mould infection by post-treatment of panels with natural extracts from fungal antagonists. In this study, the culture metabolites of a fungal antagonist, Phaeotheca dimorphospora DesRochers & Ouellette, were extracted, and the antibiotic activity of the extracts was tested in Petri plates against various moulds and decay fungi. The OSB panels were then dip-treated with the extracts and exposed to a humid environment for mould growth testing in a period of 8 weeks. The results showed that the mycelia growth of all fungi tested (moulds, white-rot and brown-rot fungi) was inhibited by the extracts of P. dimorphospora on agar plates. Panel samples dipped with the fungal extracts in acetone got little mould growth on them, whereas untreated control panels and those samples treated with acetone alone were seriously affected by various moulds.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

Microbial response to drought in a Texas highplains shortgrass prairie

The population of the microbial flora of a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) prairie near Amarillo, Texas, was studied during 1971 after a severe drought. Bacteria, fungi, and algae were estimated by plate count and terminal dilution procedures. Rates of grass and paper decomposition were determined. The microbial flora of soil associated with bovine-grazed grass did not differ significantly from the flora associated with ungrazed grass, either qualitatively or quantitatively. During drought, a greater number of fungi were found in soil associated with prickly pear than in that associated with blue gramma grass. The microbial biomass decreased one full log between the surface and a depth of 50 cm, and the percentage of anaerobes increased with depth. The maximum numbers of fungi and algae detected were 8 × 10⁵ and 6 × 10⁴/g respectively. A linear relationship existed between the microbial biomass and soil moisture. The maximum number of aerobic, heterotrophic bacteria detected was 1.5 × 10⁸ viable cells per g of soil.     

Do the unique properties of nanometals affect leachability or efficacy against fungi and termites?

Nanotechnology has the potential to affect the field of wood preservation through the creation of new and unique metal biocides with improved properties. This study evaluated leachability and efficacy of southern yellow pine wood treated with copper, zinc, or boron nanoparticles against mould fungi, decay fungi, and Eastern subterranean termites. Results showed that nanocopper with and without surfactant, nanozinc, and nanozinc plus silver with surfactant resisted leaching compared with metal oxide controls. Nearly all nanoboron and boric acid was released from the treated wood specimens during leaching. Mould fungi were moderately inhibited by nanozinc oxide with surfactant, but the other nanometal preparations did not significantly inhibit mould fungi. Mass loss from Gloeophyllum trabeum was significantly inhibited by all copper preparations, while Antrodia sp. was not inhibited by nanometal treatments. Nanometals imparted high resistance in southern yellow pine to the white-rot fungus, Trametes versicolor. Unleached specimens treated with nanoboron or nanozinc plus surfactant caused 100% and 31% mortality, respectively. All specimens treated with nanozinc or nanozinc plus silver inhibited termite feeding, but the copper treatments were less effective against termites. Nanozinc possessed the most favorable properties: leach resistance, termite mortality, and inhibition of termite feeding and decay by the white-rot fungus.     

New antifungal agents that inhibit the growth of Candida Species: Dichlorinated 8-quinolinols

Five dichlorinated 8-quinolinols (2,5- 5,6-, 3,5-, 3,7-, and 4,5-dichloro-8-quinolinol) were tested against Candida albicans and C. Tropicalis in Sabouraud dextrosebroth with and without bovine serum. The 5,6-, 3,5-, and 3,7-dichloro-8-quinolinols proved to be more effective than the control, 5-fluorocytosine. In cytotoxicity tests employing baby hamster kidney (BHK) cells, all test agents proved to be more cytotoxic than the control. However, the minimum inhibitory concentration (MIC) of 3,5-dichloro-8-quinolinol to both fungi was only one tenth the cytotoxic dose,suggesting that the compound may be useful as a topical or systemic antifungal agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Determining the Suitability of Lactobacilli Antifungal Metabolites for Inhibiting Mould Growth

Summary In recent years, public concern about indoor mould growth has increased dramatically in the United States. In this study, lactic acid bacteria (LAB), which are known to produce antimicrobial compounds important in the biopreservation of food, were evaluated to determine if the same antimicrobial properties can be used to inhibit mould fungi that typically colonize wood. Based on biomass measurement, cell-free supernatants from Lactobacillus casei subsp. rhamnosus and Lactobacillus acidophilus grown in deMan Rogosa Sharpe (MRS) broth inhibited 95–100% growth of three mould fungi and one stain fungus associated with wood-based building materials. Lactic acid and four unknown compounds ⩽ kDa molecular weight were fractionated from the culture supernatant by thin layer chromatography and high-performance liquid chromatography. Antifungal activity, which was attributed to one or more unknown metabolites, was retained during heating and neutralization. A 1:2 dilution of L. casei supernatant inhibited 100% growth of all test fungi.     

Salmonella typhimurium poultry isolate growth response to propionic acid and sodium propionate under aerobic and anaerobic conditions

Propionic acid and its sodium salt have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acids in poultry feed may have a potential role in inhibiting growth of Salmonella in the chicken intestine. In this study, we determined growth response of a Salmonella typhimurium poultry isolate to propionic acid and sodium propionate under aerobic and anaerobic conditions. Growth rate consistently decreased with the addition of greater concentrations of either propionic acid or sodium propionate. The extent of growth inhibition was much greater with propionic acid than the sodium form. Media pH decreased only with addition of propionic acid. Growth inhibition was more effective under anaerobic growth conditions with either propionic acid or sodium propionate. When determined at the same pH level, growth rate was significantly lowered by addition of 25 mM of either propionate or sodium propionate alone, and also by the decrease in pH levels (P<0.05). These results showed that growth inhibition of S. typhimurium by propionic acid or sodium propionate is greatly enhanced by pH decrease, and to lesser extent by anaerobiosis. We also found that sodium propionate was more inhibitory for growth of S. typhimurium than propionic acid when compared at the same pH levels.     

High production of D-β-hydroxyisobutyric acid from methacrylic acid by Candida rugosa and its mutant

Production of D-β-hydroxyisobutyric acid (D-HIBA) from methacrylic acid (MA) was investigated using Candida rugosa IFO 0750 and its mutant. Cell growth decreased as the MA concentration increased and was inhibited at D-HIBA concentrations higher than 30 g/l. Optimal MA concentration for D-HIBA production was in the range of 10–20 g/l. It was also noted that cell growth and D-HIBA production were inhibited by higher concen-trations of Na⁺, K⁺, and NH₄ ⁺, which were required for pH control during cultivation. With a suitably designed feeding mode of MA, the parent strain produced 65 g/l of D-HIBA after 120?h of fed-batch cultivation, but molar conversion yield of D-HIBA was less than 40%. The mutant, unable to assimilate propionic acid, produced as high as 70 g/l of D-HIBA in the same culture period with a molar conversion yield of more than 70%.