Conidium "fitness" in Trichoderma

A major constraint for use of biocontrol agents (BCAs) is inconsistent performance under changeable environmental conditions. This study aimed to develop new knowledge of effects of growth conditions, to increase persistence and efficiency of the key agent Trichoderma atroviride LU132, and to understand the factors which may influence conidium “fitness” for biocontrol formulations of conidia, that are robust (long-surviving) and active against target plant pathogens. Effects of culture conditions, (incubation period, temperature, nutrients, water activity and pH) productivity, germinability and bioactivity of T. atroviride LU132 conidia were assessed, in assays against the soil-borne plant pathogen, Rhizoctonia solani. Conidium fitness was assessed after storage and in glasshouse pot experiments. Biochemical and ultrastructural characteristics of conidia produced in different culture conditions were also examined, in relation to conidium fitness. The influence of incubation temperatures (20, 25, or 30°C) on the production of conidia was assessed under constant light over a 25 d period. Two measures of quality of the resulting conidia were also determined; - germination and subsequent bioactivity against Rhizoctonia solani. Maximum conidium production occurred at 25°C after 20 d but was less at 25 d. Conidia produced at 30°C germinated more rapidly and gave the greatest bioactivity against R. solani in comparison with incubation at 20 or 25°C. An incubation period of 25 d gave the greatest bioactivity compared with shorter incubation periods. To examine the effects of extending incubation time on conidium production, germination and bioactivity, the experiment was extended at 25°C for up to 50 d, which resulted in a second peak of conidium production at 45-50 d. These conidia had optimum germination after 20 and 25 d incubation, and optimum bioactivity was achieved with conidia harvested after 15 d. Therefore, temperatures near 25°C and incubation period of 15 d were shown to be optimum for production of T. atroviride LU132. Formulations of T. atroviride based on optimised production of conidia may not result in optimal bioactivity. This is the first report indicating that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Conidium production of this biocontrol strain was shown to be a continuous process, and a scheduled dark/light regime increased conidium production. Furthermore, conidium production is likely to be on 20 d base cycle, which is probably dependent on colony age rather than abiotic factors. This is also the first report of bimodal conidium production in a Trichoderma biological control agent. Identification of the production and storage factors that affect conidium fitness can assist the success of biological control agents. Conidia from the culturing regimes which resulted in greatest and least bioactivity against R. solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Conidia were examined after storage at 30˚C and at 0 or 50% relative humidity (RH) over six months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH. The greatest number of conidia and greatest germination resulted from C to N ratios of 5:1 or 160:1, amended with sucrose at 25°C, but greatest bioactivity resulted from conidia produced at 30˚C. However, fewer conidia were produced at 30˚C, and the least germination and bioactivity resulted from conidia produced at 20°C, both amended with dextrose. The conidia adapted to high temperature of 30°C (amended with dextrose) or nourished at C to N ratio of 5:1 (amended with sucrose) showed the greatest conidium fitness. Further experiments assessed effects of temperature and hydrocarbon type. Interactions of temperatures (20 or 30°C) vs sugars (dextrose, 4.2 g/L or sucrose, 4.2 g/L in constant C:N ratios of 5:1) were examined for bioactivity and colonisation potential in pot experiments with ryegrass in the presence of R. solani. Conidia produced at 20°C with dextrose (4.2 g/L in constant C:N ratio of 5:1) gave the greatest bioactivity, where rhizosphere and bulk soil assessments were carried out. The bimodal population cycle in T. atroviride LU132 recurred in pot experiments (recorded as colony forming unit (CFU)) in a manner similar to that observed in agar plates, but in an approx. 15 d cycle, indicating that simulated natural conditions shortened the Trichoderma life cycle. Biochemical and ultrastructural studies were carried out to determine relationships between quality variations and cellular characteristics for conidia produced in different culturing conditions. The effect of culture conditions on trehalose accumulation was most marked, while differences in arabitol and mannitol were much less. The least trehalose accumulation was detected in conidia produced at 20°C (13 mg/g dry conidia). This could justify the least conidium survival and bioactivity during storage. Fatty acids detected in conidia by gas chromathography were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1 c9) and linoleic acid (18:2 c9, 12). Linoleic acid was the most abundant (overall mean of 26%), and stearic acid was the least abundant (8%). The conidium production treatment at 20°C gave the greatest amount of fatty acids (66 µg/g of dry conidia), giving conidia deep dormancy or other deterioration effects, while the C:N 5:1 treatment which gave high bioactivity after storage gave the least conidium fatty acid content (12 µg/g dry conidia). Ultrastructural differences of conidia were linked to differences in conidium survival and successful biocontrol establishment. Low electron density of conidium contents and accumulation of lipid droplets were associated with less integrity and viability. Conidia produced at 20°C showed significant disorganisation of cellular structures. This research has provided new insights which can form the basis of efficient production of Trichoderma-based biocontrol agents. Additional insights into the basis of conidium fitness in T. atroviride LU132 have also been provided

Effects of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride

Aims: The goal was to determine the effect of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride LU132 against Rhizoctonia solani. Methods and Results: The incubation temperatures of 20, 25 or 30°C were assessed on the production of T. atroviride conidia under constant light over a 25 and 50 days periods. The resulting conidia were also studied for germination and bioactivity. Conidium production was maximum at 25°C after 20 days. The second peak of conidium production occurred at 45-50 days. Incubation at 25°C after 15 days showed optimum production of T. atroviride LU132. Conidia produced at 30°C gave the greatest germination and bioactivity in comparison with incubation at 20 or 25°C. Conclusion: This study indicates that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Formulations based on production of the high conidia yield may not result in optimal bioactivity and there is a trade-off between quantity and quality of T. atroviride LU132 conidia. Conidium production was shown to be a continuous process, and increased under a dark/light regime. This is the first report of bimodal conidium production in a Trichoderma biological control agent (BCA), which is likely to be on 20 days cycle, and is dependent on colony age rather than abiotic factors. Conidia produced after 15 days are likely to be the most suitable for use in commercial production of this strain as a BCA. Significance and Impact of the Study: Most studies on Trichoderma-based BCA have only shown the effect of culture conditions on the high conidia yield regardless of conidium quality. This study is the first report on conidium quality affected by principal culture conditions for Trichoderma biological control formulations

Bioactivity, biochemical and ultrastructural characteristics of Trichoderma atroviride conidia influenced by different culturing conditions: factors affecting biocontrol potential

Identification of production and storage factors that affect conidial germination and bioactivity (fitness) will assist the success of biological control agents. Effects of abiotic factors (temperature, nutrients, water activity, pH) on conidial fitness of Trichoderma atroviride LU132 during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidial fitness over time. Fitness of the test conidia was examined after storage at 30˚C and at 0 or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH. The greatest number of conidia and greatest germination resulted from growth at 25°C, but greatest bioactivity resulted from conidia produced at 30˚C. Different C to N ratios (5:1 or 160:1) did not affect these parameters. However, fewer conidia were produced at 30˚C, and the least germination and bioactivity resulted from conidia produced at 20°C. The different environmental factors were not independent. For example, conidial production at 30°C is probably accompanied by water stress, oxidation, and rapid pH change which may also affected fitness. Further study was performed based on temperature and hydrocarbon type. Biochemical and ultrastructural studies were carried out to determine relationships between quality variations and cellular characteristics for conidia produced in different culturing conditions. Results of fatty acid, and sugar (trehalose and polyols) analyses, and ultrastructural observations are presented in this paper

Homogeneity Distance Classification Algorithm (HDCA): A Novel Algorithm for Satellite Image Classification

Image classification is one of the most common methods of information extraction from satellite images. In this paper, a novel algorithm for image classification based on gravity theory was developed, which was called “homogeneity distance classification algorithm (HDCA)”. The proposed HDCA used texture and spectral information for classifying images in two iterative supplementary computing stages: (1) merging, (2) traveling and escaping operators. The HDCA was equipped by a new concept of distance, the weighted Manhattan distance (WMD). Moreover, an improved gravitational search algorithm (IGSA) was applied for selecting features and determining optimal feature space scale in HDCA. In the case of multispectral satellite image classification, the proposed method was compared with two well-known classification methods, Maximum Likelihood classifier (MLC) and Support Vector Machine (SVM). The results of the comparison indicated that overall accuracy values for HDCA, MLC, and SVM are 95.99, 93.15, and 95.00, respectively. Furthermore, the proposed HDCA method was also used for classifying hyperspectral reference datasets (Indian Pines, Salinas and Salinas-A scene). The classification results indicated substantial improvement over previous algorithms and studies by 2% in Indian Pines dataset, 0.7% in the Salinas dataset and 1.2% in the Salinas-A scene. These experimental results demonstrate that the proposed algorithm can classify both multispectral and hyperspectral remote sensing images with reliable accuracy because this algorithm uses the WMD in the classification process and the IGSA to select automatically optimal features for image classification based on spectral and texture information

Understanding the survival, persistence, and bioactivity of the biocontrol agent Trichoderma atroviride LU132

Identification of the production and storage factors that affect conidial germination and bioactivity (fitness) will assist the success of biological control agents. Abiotic factors (temperature, nutrients, water activity, pH) during production were studied and then the effects of culturing conditiond on conidial fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time

Germination and bioactivity of Trichoderma atroviride affected by culturing and storage conditions

Identification of the production and storage factors that affect conidial germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidial fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity, pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage condition on conidial fitness. Fitness of the test conidia was examined after storage at 30°C and at 0 or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH. The greatest number of conidia and germination percentage resulted from conidia produced at 25°C, but greatest bioactivity resulted from those produced at 30°C. Different C to N ratios (5:1 or 160:1) did not affect these parameters. However, fewer conidia were produced at 30°C, and the least germination and bioactivity resulted from conidia produced at 20°C. Conidia can be divided into two groups: those adapted to extreme culturing conditions (e.g. high temperature), and those protected by nutrients during storage. However, environmental factors are not independent. For example, conidial production at 30°C is probably accompanied by water stress, oxidation, and rapid pH change which may also affect fitness

Quantitative and Qualitative Assessment of Arak city Hospital Wastewater on Sewage Network and Treatment Plant of Arak City, (from Point of Organic Pollutants and Heavy Metals)

Background: In this study quality and quantity characteristics wastewater of hospitals, clinics and health centers of Arak city and the potential impacts of them on Arak wastewater plant investigated. Methods: In this cross-sectional  study, which done during 2011-2012, the quantity and quality  of wastewater via point of COD,BOD5, pH, TKN, PO4and also heavy metals in the hospitals, clinics and health center of Arak were studied. Then, the effects of these pollutants as equal to person on wastewater convey system and wastewater treatment plant were assisted. Results: Monitoring of BOD5, TKN, and PO4 indicated that the daily disposal rate of these pollutants were equal 778.14, 102.7, and 53.6 kg/d, respectively, which equal to 15763, 51351, and 6700 person, respectively. The average water consumption of these centers was equal to 8.2l/s, and the estimated measure of produced wastewater was6.14 l/s. Also, after analysis the measure of heavy metals, Hg production (99.331 g/d) was more than other heavy metals, and the next rank was related to Zn (41.96 g/d) and Ag (41.96g/d), respectively. Conclusion: Although existence pretreatment process units can help to decrease the side effects of produced wastewater, this kind of wastewater needs complete treatment. Discharge of this kind wastewater to absorption trenches led to adverse health impacts in future. Therefore, a construction separate wastewater treatment plants and proper operation of these systems can reduce ecosystem impacts of wastewater discharges

Biological fitness of <i>Trichoderma atroviride</i> during long-term storage, after production in different culture conditions

<div><p>ABSTRACT</p><p>Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of <i>Trichoderma atroviride</i> LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against <i>Rhizoctonia solani</i> in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C : N 5 : 1 and C : N 160 : 1, indicating that C : N ratio in culture medium is not important for conidium survival of <i>T. atroviride</i>.</p></div

Biochemical characteristics of <i>Trichoderma atroviride</i> associated with conidium fitness for biological control

<div><p>ABSTRACT</p><p>Effects of abiotic factors during production (temperature, nutrients, water activity, pH) on conidium fitness (quantity and quality) of <i>Trichoderma atroviride</i> LU132 (a key biocontrol agent) were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against <i>Rhizoctonia solani</i> in dual culture assays were selected to assess effects of storage conditions on conidial fitness over time. Further studies assessed interaction effects of temperatures (20°C or 30°C) and sugars (dextrose or sucrose) on conidium germination and bioactivity as fresh conidia, or after 6 months of storage. Biochemical analyses of sugars and fatty acids were carried out to determine relationships between quality variations and cellular characteristics for conidia produced in different culturing conditions. Low trehalose content in conidia (e.g. at 20°C) was associated with the least conidium fitness, although high trehalose content did not necessarily support conidium fitness. High proportions of total fatty acids in conidia were mostly associated with the least conidium fitness. When <i>Trichoderma</i> was grown at high carbon to nitrogen ratio (e.g. at C:N 160:1), the total conidium fatty acids content increased. This study also indicated that the monosaccharide dextrose is metabolically optimal for <i>T. atroviride</i> LU132 at 20°C while the disaccharide sucrose is optimal at 30°C. These studies indicate that physical growth conditions and nutritional requirements attribute in conidium fitness of <i>T. atroviride</i> LU132, and provide important knowledge supporting optimum production of biocontrol agents based on <i>T. atroviride</i>, and possibly other similar biocontrol agents.</p></div

Endophytic colonisation of perennial ryegrass by Trichoderma atroviride

Trichoderma spp. are widely used as biocontrol agents and plant growth promoters. Endophytic colonisation of perennial ryegrass (Lolium perenne) by T. atroviride strains was studied in laboratory, greenhouse and field experiments. Four strains of T. atroviride were inoculated into in vitro agar cultures or potting mix with ryegrass seeds for colonisation studies. The strains were also produced as prill or granule formulations for application in the field experiment. Microscopy was used to observe fungal structures in plant tissue segments from pot- or field-grown plants. Segments were also surface sterilised and placed onto Trichoderma selective media. Fungal colonies recovered were verified as T. atroviride by sequencing the tef1 gene. No endophytic colonisation occurred in any non-inoculated plants, and no Trichoderma endophytic colonisation was observed in field-grown plants. However, microscopy revealed fungal hyphae and reproductive structures characteristic of Trichoderma in root and stem sheath tissues of inoculated plants from in vitro cultures and pots. These results were verified by sequencing the tef1 gene. This study has demonstrated endophytic colonisation of ryegrass by T. atroviride strains, which may be related to beneficial effects on plant growth and disease control