Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

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.     

Outdoor Studies of the Efficacy of Alternaria alternata in Controlling Amaranthus retroflexus

Application of 250 L ha -1 containing 10 7 Alternaria alternata conidia ml -1 caused 61% reduction in dry weight and a 45% mortality in Amaranthus retroflexus plants growing in a wheat crop. However, only 10-22% of the applied conidia were retained on the leaf. In one experiment, conidia remained viable for 4 days on the leaf surface until conditions were favourable for germination. Competition from the wheat crop, as well as its creation of a moist micro-climate, improved the control of Am. retroflexus by A. alternata . Total control of Am. retroflexus may be difficult to achieve with A. alternata , but suppression to reduce or eliminate its competitive effect on crops is possible.     

Hydroponic Seedling Bioassay for the Bioherbicides Colletotrichum truncatum and Alternaria cassiae

A rapid bioassay was developed to measure the bioherbicidal efficacy of spore preparations of the pathogens Colletotrichum truncatum (Schwein.) Andrus and W. D. Moore and Alternaria cassiae Jurair and Khan on hemp sesbania (Sesbania exaltata) and sicklepod (Cassia obtusifolia), respectively. The system uses 4-day-old dark-grown seedlings (grown hydroponically in paper towel cylinders) which were sprayed with spore suspensions. Shoot lengths were monitored non-destructively, and recorded over time under conditions of dark growth, 90-100% relative humidity and 25 C. Shoot growth inhibition and stem collapse (mortality) were directly related to the spore concentration applied. Generally, at 10 3 - 10 4 spores ml-1, these pathogens caused significant shoot growth inhibition within 25-30 h and seedling death within 40-50 h. This bioassay has been used to study herbicide-pathogen interactions, and may be extended to determine the bioherbicidal efficacy of different pathogen isolates, pathovars or spore formulations. This technique is more rapid, uses a lower inoculum volume, requires less space and is performed under more controlled conditions than conventional greenhouse bioassay methods. The data obtained are more quantitative than those obtained from bioassays relying on visual rating systems.     

Effects of Storage on Viability and Efficacy of Granular Formulations of the Microbial Herbicides Alternaria alternata and Trematophoma lignicola

Alternaria alternata Pesta (gluten matrix) granules stored at 12% relative humidity were still viable and infective, giving total control of Amaranthus retroflexus after 24 months storage. Viability of the propagule in the granule was an important factor of the evaluation test and not just the viability of the granule. The concentration of inoculum in the granule was important, with 10 5 to 10 6 conidia g -1 being the ideal, since lower concentrations may not be able to out-compete the soil micro-flora. Even with soil application, the effect of local humidity was still important. Trematophoma lignicola was also formulated as microbial herbicide granules but the conidia were severely damaged in the production process.     

Investigation of Spray Application of Microbial Herbicides Using Alternaria alternata on Amaranthus retroflexus

Inclusion of Alternaria alternata conidia in a spray formulation affected the distribution pattern on the target. The dry weight of Amaranthus retroflexus plants was reduced by more than 83% when A. alternata conidia (107 ml -1 ) were applied at 200 L ha -1 or greater and when given a 24 h dew period. At low application volumes (25 or 50 L ha -1 ) plant dry weight was reduced by only 29 or 54%. After 7-8 h dew period, conidial germination on the leaf surface was 11-19%. This increased to 62-91% after 24 h dew period. Counts of conidia on leaves indicated that up to 86% of the conidia sprayed were not retained on the target plant, or did not reach it. This is reflected in lesion numbers per unit area being only 3-5% of the calculated theoretical numbers. The results cast doubt on the suitability of A. alternata as a microbial herbicide for the control of Am. retroflexus .     

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.     

L型氨基酸对烟草赤星病菌毒素毒性的影响

为确定不同氨基酸对烟草赤星病菌产生的毒素毒性的影响,作者采用摩擦接种处理法、浇根处理法、种子根处理法三种生物测定方法,确定了以烟草种子根处理法作为烟草赤星病菌的毒性测定方法。用12种氨基酸分别以5种不同浓度与AT毒素混合接种,根长测定结果表明：L-谷氨酰胺,L-天冬酰胺,L-组氨酸所有供试浓度均增强病原菌毒素的毒性,而其余9种L型氨基酸对烟草赤星病菌GL-6产生的毒素有抑制作用,其中L-甲硫氨酸、L-亮氨酸、L-赖氨酸适宜浓度可明显降低供试病原菌毒素对烟草的致病性,与对照差异极显著,其有效浓度分别为0．02mg／ml、0．02mg／ml、0．02～2mg／ml。     

柠檬醛熏蒸对互隔交链孢生长及其产毒的抑制作用

互隔交链孢是一种重要的能产生交链孢酚（AOH）等真菌毒素的植物病原菌。精油是重要的抑制病原菌侵染的挥发性植物提取物,其活性组分包括柠檬醛等。本研究表明柠檬醛可高效地抑制互隔交链孢的生长和AOH毒素的产生。柠檬醛熏蒸能够引起互隔交链孢菌丝断裂影响其延伸,而对其分生孢子结构的影响不明显。柠檬醛能够引起互隔交链孢活性氧生成的紊乱,这可能是导致AOH显著下降的原因之一。由于柠檬醛能高效抑制互隔交链孢生长和产毒,因此其可作为传统熏蒸剂的潜在替代品,以防控互隔交链孢引起的病害以及毒素污染。柠檬醛抑制互隔交链孢生长产毒的研究为其开发与应用奠定了良好的基础。     

Factors Influencing the Efficacy of the Potential Microbial Herbicide Alternaria alternata (Fr.) Keissler on Amaranthus retroflexus (L.)

Alternaria alternata , applied to run-off at 107 conidia ml -1 and given a 6 h dew period, caused an 88.5% reduction in dry weight of 4 to 5 true-leaf plants of Amaranthus retroflexus . This level of control was achieved whether the inoculated plants were grown in the glasshouse or outside. Formulation of the pathogen in oilseed rape emulsion reduced its dew period requirement from 24 h or more to 8 h or less. For example, with 106 conidia ml -1 , equivalent effects were found with 8 h dew with emulsion formulation compared to 48 h dew with the Tween formulation. Allowing spray deposits to dry on inoculated plants for ca 7 h, before exposing to dew increased the efficacy of the pathogen.     

Heat-stable Proteins and Acquisition of Desiccation Tolerance in Peanut Seeds

The acquisition and induction of desiccation tolerance associated with the expression of heat-stable proteins in the developing peanut (Arachis hypogaea L. ) seeds were studied. Desiccation tolerance of peanut seeds was achieved during 45 to 65 DAP (days after pegging) embryogenesis, while a set of low molecular weight (9 to 15.5 kD) heat-stable polypeptides was preferentially expressed. Slow drying regime applied in vitro to 25 and 35 DAP peanut embryos induced desiccation tolerance and the expression of the same subset of polypeptides. Mature drying treatment enhanced the ability of 65 DAP peanut embryos to withstand fast drying, also increased the heat stability of arachins, the major peanut storage protein, which was heat labile during 45 to 65 DAP embryogenesis. It was concluded that the heat-stable proteins may contribute to desiccation tolerance of the peanut seeds, and the low molecular weight heat-stable polypeptides may confer nonspecifieally heat tolerance on peanut storage proteins which were normally heat labile.     

Effects of Alternaria alternata f.sp. lycopersici toxins on pollen

Summary Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.     

Deleterious effect of herbicides on waterhyacinth biocontrol agents Neochetina bruchi and Alternaria alternata

The integration of biological and herbicidal methods is advocated to manage waterhyacinth, but this can only be achieved when herbicides are nontoxic to the biocontrol agent. Therefore, laboratory experiments were conducted to determine the toxic effect of herbicides on the insect biocontrol agent, the waterhyacinth weevil, Neochetina bruchi Hustache, and phytopathogen, Alternaria alternata, with two commonly used herbicides, glyphosate and 2,4-dichlorophenoxy acetic acid at three recommended doses. The herbicides were sprayed on the waterhyacinth weevils and added to the nutrient media of A. alternata. 2,4-D caused higher weevil mortality (6.7, 13.3 and 15.6%) as compared to glyphosate (3.3, 5.6 and 11.1%), at three doses over 72 h. There was also a decrease in feeding in the herbicide treated leaves. When the weevils were allowed to move freely between the herbicide treated and untreated plants, higher orientation of the weevils was found on the untreated waterhyacinth than on the treated ones. Neither of the two herbicides actually killed the fungus but both inhibited its growth. Glyphosate though, delayed mycelial growth yet stimulated sporulation while 2,4-D inhibited both growth and sporulation. Glyphosate at low concentration did not affect the virulence of A. alternata, while fungi grown on 2,4-D amended plates lost their virulence.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

链格孢菌粗毒素对菊花‘神马’幼苗生长及生理代谢的影响

由链格孢菌引起的菊花黑斑病严重降低了菊花的品质和产量.链格孢菌在代谢过程中分泌的粗毒素是菊花黑斑病发生的主要致病因子之一.本文从菊花黑斑病发病叶片中分离筛选出致病真菌链格孢菌1株,研究其粗毒素对菊花幼苗‘神马’生长的影响以及测定盆栽幼苗叶片细胞膜相对透性、抗性物质含量、诱导酶活性及代谢物质含量变化.结果表明: 链格孢菌粗毒素对菊花‘神马’幼苗的株高、茎粗、根长均有抑制作用,毒素浓度与抑制效果呈正相关,且粗毒素原液处理14 d后,菊花幼苗株高、茎粗、根长受到显著抑制,分别比对照减少了28.9%、21.4%和23.3%;链格孢菌粗毒素处理菊花‘神马’幼苗后,根系组织细胞膜透性随着毒素浓度的增加而增加,在同一毒素浓度处理下,菊花幼苗叶片细胞膜透性随着处理时间的增加呈先增大后降低的趋势;毒素原液处理菊花幼苗后,菊花幼苗叶片中抗性物质可溶性蛋白、丙二醛(MDA)以及脯氨酸含量均显著提高.链格孢菌10倍稀释液处理对叶片苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)活性的提高最为显著.链格孢菌粗毒素对切花菊‘神马’幼苗的致病作用主要通过抑制菊花幼苗根、茎的正常生长,增加菊花幼苗叶片细胞膜透性,影响切花菊幼苗叶片中抗性物质代谢以及提高叶片保护酶活性而影响植株正常生理代谢.     

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.     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

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.     

Biocontrol potential of five Burkholderia and Pseudomonas strains against Colletotrichum truncatum infecting chilli pepper

The effect of bacterial antagonism against the causal agent, Colletotrichum truncatum was assessed as a potential alternative in managing anthracnose in chilli pepper. Out of 104 contrasting bacterial colonies isolated from rhizosphere soil of a forest floor, five isolates caused the radial growth inhibition greater than 90% (Significant at p?<?0.05 level) of C. truncatum in dual cultures. Based on 16S rRNA analysis, these antagonistic bacterial isolates were identified as Pseudomonas aeruginosa, Burkholderia arboris, Burkholderia gladioli and Burkholderia rinojensis. The selected promising antagonists showed nearly 100% inhibition of the spore germination of C. truncatum in vitro. These antagonists produced antifungal compounds which are diffusible in nature. Microscopic studies of blackened fungal hyphae, which were subjected to antagonism showed many deformations such as thickening, swelling and malformation. In vivo study revealed that C. truncatum inoculated chilli pepper seeds treated with the five antagonists significantly inhibited the incidence of seed colonisation (p?<?0.05) by the pathogen. At the post emergence stage, the survival percentages and vigour indices of all the antagonists’ treatments, except B. rinojensis strain 1, were significantly higher compared to the untreated control. The efficacy of the selected antagonists in managing anthracnose fruit rot was 100% at the colour breaking stage of chilli pepper fruits. These bacterial antagonists had a negative effect on C. truncatum spore attachment and subsequent colonisation on chilli pepper leaves except in the treatment of B. arboris. The results of in-vitro and in-vivo studies, suggest that the screened antagonistic bacterial isolates are potential biocontrol agents and need to be further studied for the biochemical basis of their activity against C. truncatum.