Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .     

Microscopic and Cytochemical Analysis of Extracellular Matrices and Endogenous Reserves of Conidia of Colletotrichum truncatum Harvested from Carbon- and Nitrogen-limited Cultures

The effects of nutrition during conidiation on extracellular matrices (ECM) and endogenous reserves of conidia of the promising mycoherbicide Colletotrichum truncatum have been examined. Transmission electron microscopy showed no ECM on the conidia, regardless of the C:N ratio (10:1, 30:1 or 80:1) of the medium on which they were produced. Cytochemical analysis using fluorochrome-labelled lectins revealed the presence of specific sugars in ECM around the germlings. Furthermore, ECM-containing amino groups (basic protein) were detected using colloidal gold. However, nutrition during conidiation had no effect on the patterns of lectin labelling or the pattern of colloidal gold staining of germlings. The relative amounts of trehalose, glycerol and mannitol in conidia produced in a liquid medium with a C:N of 10:1 were more than those obtained when the C:N was 30:1 or 80:1. Thus, internal carbohydrates such as trehalose and polyols may play an important role in viability of conidia during long-term storage.     

Water Activity and Other Factors that Affect the Viability of Colletotrichum truncatum Conidia in Wheat Flour-Kaolin Granules ('Pesta')

Optimization of shelf-life is critically important for biocontrol products containing living microorganisms. Conidia of Colletotrichum truncatum, a fungal pathogen of the weed, hemp sesbania (Sesbania exaltata), were produced in shake flasks (corn meal-soya flour medium) and on Emerson Yp Ss agar and formulated in wheat flour-kaolin granules ('Pesta'). The granules were conditioned at water activities of 0, 0.12, 0.33, 0.53 and 0.75 during storage at 25 C over desiccant or saturated salt solutions. The longest shelf-life (conidial inoculum viability) was found in samples in the water activity range 0-0.33, where the water was bound by the matrix and not readily available to the fungus. At a water activity of 0.12, granules were 100% viable (on water agar) for at least 24 weeks, and were 87% viable after 1 year. Sucrose (5% w/w) partially counteracted the detrimental effect of high water activity on the shelf-life of C. truncatum when incorporated in the granules.     

Solid Substrate Formulations of the Mycoherbicide Colletotrichum truncatum for Hemp Sesbania ( Sesbania exaltata ) Control

Colletotrichum truncatum was grown on kernels of eight different grains for 3 or 4 weeks at room temperature (22-24°C). Fresh preparations of conidia as well as fungus-infested corn and rice suspensions resulted in 100% mortality of hemp sesbania seedlings when sprayed postemergence with a 14 h dew period. Fresh preparations of mycelia and fungus-infested sorghum suspensions resulted in 90 and 65% mortality of hemp sesbania seedlings, respectively. Lower mortality ( ≤15%) occurred with the other ground fungus-infested grain suspensions. Fresh preparations of conidia, fungus-infested corn, rice and sorghum, and mycelia, when applied to soil pre-emergence, resulted in 100, 94, 100, 83 and 71% mortality of hemp sesbania seedlings 14 days after application, respectively. Lower mortality ( ≤23%) occurred with the other ground fungus-infested grain preparations. Freshly-prepared C. truncatum at 6.25, 12.5, 25 and 50 mg fungus-formulated rice cm -2 of soil surface, applied pre-emergence or at the time of planting, killed 97, 100, 100 and 100% of hemp sesbania, respectively. After storage at 22-24°C for 6 to 24 months, the rice formulation caused 67 to 93% mortality after 6 months, 39 to 81% after 12 months, and ≤2% after 24 months, respectively. When C. truncatum was refrigerated at 4-6°C, the rice formulation retained good efficacy through 24 months, and when frozen, for up to 8 years. C. truncatum formulated on rice stored under all the above conditions contained mainly sclerotia at 2.4 x 10 5 sclerotia g -1 . C. truncatum killed hemp sesbania seedlings with a single soil application through 4 plantings on the same soil. These results indicate that rice and possibly corn are excellent solid substrates for the formulation of C. truncatum . This is a simple and effective method for enhancing the activity of C. truncatum against hemp sesbania.     

Tolerance of thermophilic and hyperthermophilic microorganisms to desiccation

We examined short- and long-term desiccation tolerance of 31 strains of thermophilic and hyperthermophilic Archaea and thermophilic phylogenetically deep-branching Bacteria. Seventeen organisms showed a significant high ability to withstand desiccation. The desiccation tolerance turned out to be species-specific and was influenced by several parameters such as storage temperature, pH, substrate or presence of oxygen. All organisms showed a higher survival rate at low storage temperatures (−20°C or below) than at room temperature. Anaerobic and microaerophilic strains are influenced negatively in their survival by the presence of oxygen during desiccation and storage. The desiccation tolerance of Sulfolobales strains is co-influenced by the pH and the substrate of the pre-culture. The distribution of desiccation tolerance in the phylogenetic tree of life is not domain specific. Surprisingly, there are dramatic differences in desiccation tolerance among organisms from the same order and even from closely related strains of the same genus. Our results show that tolerance of vegetative cells to desiccation is a common phenomenon of thermophilic and hyperthermophilic microorganisms although they originated from quite different non-arid habitats like boiling acidic springs or black smoker chimneys.     

Studies on Desiccation and Storage of Mango Seeds

Mango (Mangifera indica L.) seed is recalitrant which taken from ripened fruits con,ained as high as 69.2'–75.5% moisture content (The moisture content of embryonic axis is 73.8–86.3%). When seeds were naturally dried for 8 days, the moisture content declined to 39.1% (in embryonic axis the moisture content declined to 46.5%) and the viability of seeds completely lost. Embryonic axis lost water slower than whole seed because of the prevention of desiccation by the large cotyledons. During natural desiccation, the conductivity of leachate increased rapidly from 2.2 μΩ·cm-1·g-1 (the same unit below) to 56.7, whereas the activities of dehydrogenase and acid phosphatase decreased drastically. When seeds were rapidly dried for 42 hours, the moisture content declined from 75.5 to 29.9%, the conductivity of leachate increased from 1.8 to 36.9 and the percentage germination changed from 100% to 10%. Desiccation damaged the cell membrane and decreased the activities of enzymes. Rapid drying was better for maintaining longevity than natural (slow) drying because the former did less damage to the cell membrane than natural (slow) drying as shown in the conductivity changes. The moisture content of excised embryonic axis decreased to 11.8% when they were dried for 8 hours by silicagel. The survival percentage of these embryonic axis was 80% when they were incubated in MS+0.2 mg/L BA+2.0 mg/L NAA+500 mg/L gln+3% sucrose+0–9% agar medium. Seeds with 51.0% moisture content (rapidly dried for l0 hours by electric fan blowing) had 65% viability after 7 months wet storage with the polyethylene bag at 15℃.     

白木香种子脱水耐性的发育变化及贮藏特性

研究白木香种子发育进程中种子性状、萌发能力和脱水耐性的变化,以及不同光温条件对种子萌发的影响和种子的贮藏特性。结果表明：白木香种子在花后78d获得最大干重,进入生理成熟期,此时萌发率接近最大值;胚在花后57～85d,脱水耐性逐渐增强,并在花后85d获得最大脱水耐性。种子萌发的适宜温度范围为25℃-35℃,光照对种子萌发有一定的抑制作用。新鲜自木香种子（含水量27．45％）在4℃低温条件下贮藏1个月后萌发率仅为30％左右,而含水量7．38％的干燥种子在4℃低温条件下贮藏120d,萌发率仍有53．33％,因此,4℃低温和适度脱水有利于种子短期贮藏。白木香种子能忍耐一定程度的脱水,但干燥至含水量7．50％以下时种子会发生损伤,因此推测白木香种子是一种中间性种子。     

Influence of Moisture Content on Temperature Tolerance and Storage of Metarhizium flavoviride Conidia in an Oil Formulation

Investigations of the temperature tolerances of Metarhizium flavoviride conidia, of different moisture contents, in an oil formulation were carried out. The conidia were dried to 5, 9, 15 or 19% moisture contents and exposed to a range of temperatures, -15, 12, 25, 38, 48 and 55 C. Viability declined due to high temperatures and high moisture contents. After 4 months of storage, the conidia were at over 80% viability in all but the 19% moisture content group of the 25 C treatment and all the samples stored at lower temperatures. In a second experiment, conidia dried to 4-5% moisture content showed greater temperature tolerance than conidia with higher moisture contents. Short-term storage under adverse conditions could be feasible with conidia dried to a suitable degree.     

Effects of Drying Rates on the Desiccation Tolerance of Citrus maxima ‘Feizhouyou’ Seeds

The effects of drying rates on the desiccation tolerance of Citrus maxima ‘Feizhouyou’ seeds at different developmental stages were studied in this paper. For seeds harvested at 130 days after anthesis (DAA), 190 DAA, 245 DAA and 275 DAA, slow dried seeds had higher desiccation tolerance than those rapid dried, with difference at significant level (P < 005). However, such improvement was little for seeds harvested at 155 DAA and 220 DAA, indicating that effect of drying rate on desiccation tolerance depends on seed developmental stages. These results accorded with previous reports on orthodox soybean seeds and maize embrys. It was suggested that the effects of drying rate on desiccation tolerance of intermediate Citrus maxima ‘Feizhouyou’ seeds mainly resulted from expression and accumulation of some desiccation related proteins induced by slow drying. On the required genetic basis, desiccation tolerance in seeds can be induced only at suitable seed developmental stages.     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

Anthracnose Caused by Colletotrichum horii on Sweet Persimmon in Korea: Dissemination of Conidia and Disease Development

Anthracnose disease caused by Colletotrichum horii (C. gloeosporioides), results in considerable economic damage to sweet persimmon in southern Korea yearly. This study deals with the life cycle of the pathogen in terms of seasonal fluctuations of spore dispersal and the development of disease based on field surveys, spore potential and fungal isolation. Anthracnose disease was observed first on twigs in the last week of May and reached an incidence of 1.2%. Subsequently, the disease increased rapidly and reached an incidence of 86% by the end of July. Infection of fruits started in mid‐June (2.8%) and increased gradually to 64.4% by the end of July. In severely infected orchards, 46.2% of diseased fruits were dropped. The pathogen began releasing conidia in the first week of May and continued until the end of September. The maximum release of spores was observed in mid‐July. To determine the optimal use of chemicals for control of anthracnose, the following spray programme was evaluated. Spraying two or three times resulted in 89.4 and 93% control, respectively, whereas spraying more than four times led to 100% control. In comparison, the disease rate of unsprayed trees was 89.8%. To control anthracnose effectively, it is recommended to take steps to eliminate inoculum sources in sweet persimmon orchards before spraying chemicals.     

CtPMK1, a mitogen‐activated‐protein kinase gene,is required for conidiation,appressorium formation,and pathogenicity of Colletotrichum truncatum on soybean

Colletotrichum truncatum, the causal agent of soybean anthracnose, invades host plants by forming a specialised infection structure called an appressorium. Mitogen‐activated protein kinase (MAPK) genes have been shown to play vital roles in several phytopathogenic fungi in regulating various infection processes, including spore germination, melanised appressorium formation, appressorial penetration and subsequent invasive growth in host plants. In this study, we identified and characterised the first Fus3/Kss1‐related MAPK gene, CtPMK1, in Colletotrichum truncatum, which is related to PMK1 in Magnaporthe oryzae. Disruption of CtPMK1 in C. truncatum resulted in a mutant with slightly reduced mycelial growth (‐30%) and melanisation that is deficient in sporulation (‐99%), as observed in the CMK1 mutant of Colletotrichum lagenarium (a synonym of Colletotrichum orbiculare, which is now the accepted name for this taxon). In contrast to CMK1 of C. lagenarium, conidia from the Ctpmk1 mutant germinated normally on glass slides and onion epidermal surfaces. Our findings suggest that there are differences in the types of in vitro functions controlled by PMK1, even between closely related species. Furthermore, the Ctpmk1 mutant failed to form appressoria or hyphopodia, subsequently resulting in the complete loss of pathogenicity on host plants. Overall, the results indicate that the Fus3/Kss1‐related MAPK gene has a conserved role in infection structure formation and pathogenicity in phytopathogenic fungi.     

炭疽病菌侵染对荔枝果实生理生化变化的影响

本研究测定了荔枝果实人工接种炭疽病菌后呼吸速率、乙烯释放量的变化和果皮氧化、过氧化作用以及与酚类代谢有关的几种酶活性的变化。结果表明,接种炭疽病菌的荔枝果实呼吸速率和乙烯释放量显著增高,果皮活性氧(O₂^·)产生速率和丙二醛(MDA)含量显著增加,超氧化物歧化酶(SOD)活性显著降低,过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性显著增高。说明炭疽病菌的侵染可导致荔枝果实呼吸速率和乙烯释放量的增高,加速荔枝果皮氧化和过氧化进程,并诱导荔枝果皮PPO、POD、PAL活性增高,是加速采收后荔枝果实衰老、褐变、腐烂的一个重要原因。     

不同脱水速率对木奶果种子脱水敏感性及抗氧化酶活性的影响

研究了脱水速率对木奶果种子脱水敏感性和抗氧化酶活性的影响。木奶果种子初始含水量高达1.72gH2O·g^-1DW,萌发率为86.67%。含水量降至0.90gH2O·g^-1DW左右时,慢速脱水种子的萌发率为97.78%,而快速脱水的种子萌发率仅为64.44%。快速脱水至含水量为0.76gH2O·g^-1DW时萌发率为21.67%,而慢速脱水至0.68gH2O·g^-1DW时,萌发率仍高达55.56%。确定了木奶果种子是对慢速脱水耐受性更高的顽拗性种子。在种子脱水过程中,相对电解质渗透速率和脂质过氧化产物（TBARs）都呈升高趋势,但慢速脱水后的种子,其TBARs升高的速率较快速脱水的慢。快速脱水的种子中超氧化岐化酶（SOD）、脱氢抗坏血酸还原酶（DHAR）和抗坏血酸过氧化物酶（APX）的活性较慢速脱水的高,而过氧化氢酶（CAT）活性较慢速脱水的低,未检测出谷胱甘肽还原酶（GR）的活性。这些结果表明,在木奶果种子脱水耐性获得过程中过氧化氢酶比其他抗氧化酶作用更大。     

Effects of Moisture Content and Temperature on Storage of Metarhizium flavoviride Conidia

The effects of moisture content and temperature on the medium-term (3-4 months) storage of conidia of Metarhizium flavoviride were investigated. Conidia harvested after 24 days of culturing on rice showed greater tolerance to long storage than conidia from 12-day cultures. The moisture content of the conidia was of greatest importance; at harvest from the culture, conidial moisture contents could be 40%, while the optimal moisture content for storage was found to be 4-5%. Dried conidia stored in oil benefited from the addition of dried silica gel, as did conidia stored as powder. A range of mineral oils proved satisfactory for storage, and when dried silica gel was added to suspensions, germination levels were 79.8% after 105 days at 28-32 C. Dried conidia stored in oil maintained germination levels of up to 96 and 85% after 80 days at 10-14 C and 28-32 C respectively. Dried conidia stored as powder retained germination levels of 95% at 10-14 C, but only up to 27% at 28-32 C. In another experiment, dried conidia maintained greater than 90% germination over 128 days, with or without silica gel at 10 - 14 C or -15 - -18 C.     

Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g⁻¹ carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 10⁸ spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L⁻¹) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L⁻¹ carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production. Received 9 December 1997/ Accepted in revised form 22 May 1998     

Seed Maturity and the Effects of Different Drying Conditions on Desiccation Tolerance and Seed Longevity in Foxglove (Digitalis purpurea L.)

The effects of different drying methods on desiccation toleranceand longevity in seeds of foxglove (Digitalis purpurea L.) wereassessed from just prior to mass maturity (when seeds have attainedmaximum dry weight), and at intervals during the post-abscissionphase of development. Tolerance of drying under seed conservationconditions (15% relative humidity, RH, and 15 °C), was acquiredclose to mass maturity at 36 d after flowering (DAF). Increasesin desiccation tolerance were induced when drying was delayedfor 4 d by placing seeds in a near-saturated atmosphere (approx.100% RH), or if seeds were pre-dried for 7 d at either approx.32% or approx. 73% RH. Irrespective of the drying treatment, seed longevity increasedthroughout the sampling period, i.e. beyond the point of massmaturity and throughout the post-abscission phase, up to thepoint of incipient natural dispersal. At each developmentalstage, delayed drying or pre-drying led to an increase in seedlongevity under controlled ageing conditions compared with seedsdried directly under seed conservation conditions. Increasesin longevity were apparent as increases in the estimates forthe intercept of transformed seed survival curves (K_i) and forthe standard deviation of the normal distribution of seed lifespans,and also in the mean time to death of individuals in storage,consistent with a continuation of ripening events. The results are discussed in relation to the assessment of seedlongevity and to current post-harvest drying practices for seedsintended for long-term ex-situ conservation.Copyright 1995,1999 Academic Press Digitalis purpurea L., foxglove, seed development, seed drying, seed longevity     

Effect of dew temperature,post-inoculation condition,and pathogen dose on suppression of scentless chamomile by Colletotrichum truncatum

Scentless chamomile, a noxious weed in western Canada, has a high natural tolerance to many herbicides. Colletotrichum truncatum, a host specific fungal pathogen, is suppressive to scentless chamomile when applied inundatively. A broadcast application was used at 200 L ha^?1 to evaluate biocontrol potential of this pathogen under a range of dew temperatures (DT), post-inoculation conditions (PIC), and pathogen doses (PD). A DT between 20 and 25°C was more conducive to infection, resulting in higher levels of disease and weed suppression as compared to 15 and 30°C. Under similar post-inoculation temperature regimes, disease was only slightly more severe in growth chambers than in the greenhouse. There was a positive linear relationship between the PD and weed suppression. An inoculum concentration >1×10⁸ spores mL^?1 reduced plant fresh weight by approximately 50% when compared to untreated controls. These results indicate that biocontrol of scentless chamomile using C. truncatum in the semi-arid Canadian prairies will likely encounter frequent non-favourable field conditions.     

Preliminary Study on Development,Germination and Desiccation Tolerance of Jatropha curcas (Euphorbiaceae)

The developmental changes in morphology and germinability of Jatropha curcas seeds, effects of temperature and light on seed germination , and changes in desiccation tolerance of mature seeds were studied in this paper. The results indicated that J. curcas seeds reached a physiological maturation at 58 days after anthesis, and that seed germination percentage reached a peak at physiological maturation, and then decreased. The optimal germination temperature was 25 to 30℃. J. curcas seeds were insensitive to dehydration at physiological maturation . There was not a notable effect of light on seed germination. Therefore, J. curcas seed was a non-photoblastic and orthodox seeds .