Ultrastructural effects of Helminthosporium maydis race T toxin on mitochondria of corn roots and protoplasts

Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.     

Activation of Clostridium botulinum Type B Toxin by an Endogenous Enzyme

It was previously postulated, based on indirect evidence, that Clostridium botulinum type B produces neurotoxin which is initially of low toxicity but which then becomes activated to highly toxic form by the action of an endogenous enzyme(s). The first direct in vitro experimental evidence in support of this hypothesis is presented here. The mildly active toxin (progenitor toxin) produced by C. botulinum type B (Lamanna) was isolated from the filtrate of a 24-hr culture and partially purified chromatographically. An enzyme that activates the progenitor toxin was also isolated from the filtrate of a 96-hr culture and purified 200-fold. The enzyme hydrolyzes synthetic substrates of trypsin but not of chymotrypsin.     

In vitro selection of yellow passion fruit genotypes for resistance to <Emphasis Type="Italic">Fusarium</Emphasis> vascular wilt

Fusarium vascular wilt (caused by Fusarium oxysporum f. sp. passiflorae) is a limiting factor in the cultivation of yellow passion fruit (Passiflora edulis). Since there is no effective and economically viable control available, development of resistant or at least tolerant cultivars are in demand. A number of procedures have been used for the initial selection of plant genotypes resistant to various fungal pathogens by means of a fungal culture filtrate or purified toxin. In this study, seeds and in vitro-grown plantlets of passion fruit were screened with different concentrations of either Fusarium oxysporum f. sp. passiflorae (FOP) culture filtrate (0, 20, 30, 40 or 50%, v/v) or fusaric acid (0.10, 0.20, 0.30 or 0.40 mM) supplemented in Murashige and Skoog (MS) basal media. Subsequently, selected plants were inoculated with a conidial suspension of FOP to assess correlation between in vivo and in vitro responses. In vitro sensitivity to the selective agents and the resistance response to the pathogen were also compared. Root growth was markedly influenced by FA, culture filtrate, and conidial suspension culture treatments. Observations indicated that roots were primary targets for attack by F. oxysporum. Successful in vitro selection of resistant genotypes by both FA and culture filtrate treatments suggested that this strategy was viable for accelerating breeding of passion fruit for resistance to the Fusarium vascular wilt.     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.     

利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究

在胡椒（Piper nigrum Linn．)茎尖丛生增殖技术的基础上,以印尼大叶种“Lampong Type”无菌实生苗作外植体源,利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择。辣椒疫霉培养滤液的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著,过滤灭菌方式可以保持辣椒疫霉培养滤液的毒性,而高温高压灭菌方式则不能。随着辣椒疫霉培养滤液浓度的增加,茎尖和丛生芽的存活率和增殖率都在下降。在存活的茎尖或丛生芽培养中,一部分可正常增殖,其余的形成愈伤组织,或者保持生长停滞的休眠状态。在选择性培养基上继代培养2次后进行生根和移栽,利用离体叶片针刺接种法对温室条件下生长的移栽植株进行抗瘟病测定。以3次抗病检测均无明显症状的植株作为抗病株。随着辣椒疫霉培养滤液浓度的增加,得到的再生植株数量降低,但其中抗病株的比例提高。利用过滤灭菌方式加入选择性培养基的处理中,25％、50％和75％的辣椒疫霉培养滤液分别获得1株、4株和3株抗病株,分别占各处理再生植株总数的1．54％、20．00％和42．86％,共获得8株,占该组处理再生植株总数的8．70％。     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

Influence of Fusarium wilt toxin(s) on carnation cells

The influence of culture filtrates of Fusarium oxysporum f.sp. dianthi which causes Fusarium wilt was investigated on growth and viability of carnation tissue cultures and leaf segments. Culture filtrates of avirulent race 1 of this fungus did not affect calli and leaf segments of cultivars both susceptible and resistant to Fusarium wilt. However, culture filtrates of virulent race 2 decreased viability and suppressed growth of callus of the susceptible cultivar. In contrast, callus of the resistant cultivar showed resistance to the culture filtrates. The results of these experiments may provide information on methods of selection of new wilt resistant carnation varieties.Abbreviations A270 absorbance at 270 nm - 2,4-d 2,4-dichlorophenoxyacetic acid - CF-MCD culture filtrate of 16064 grown in MCD medium - MCD medium modified Czapeck-Dox medium - MS medium basal medium of Murashige and Skoog - MW molecular weight - PD medium potato dextrose medium - TTC 2,3,5-triphenyl tetrazolium chloride     

Differential Responses of Guayule (Parthenium argentatum Gray) Genotypes to Culture Filtrate and Toxin from Macrophomina phaseolina (Tassi) Goidanich

The potential for using cell-free culture filtrate (CFCF) and toxin (phaseolinone) from Macrophomina phaseolina for rapid and effective screening procedures for charcoalrot resistance in guayule (Parthenium argentatum) germplasm was assessed. The CFCF and partially purified phaseolinone were incorporated into modified Murashige and Skoog solid medium aat the rates of 0-100% (v/v) and 0-1000 μg ml^-1 respectively. The medium pH was adjusted to 5.8 before solidifying with 0.8% agar. Fourweek-old seedlings of 10 guayule genotypes were plantanted in the medium, incubated and rated for phytotoxic symptoms and tissue damage over a 15-day period. In a green-house study, seedling growth, phytotoxicity and damage severity were compared in 12-week-old guayule seedlings root-inoculated with M. phaseolina microsclerotia. There were significant differences (P = 0.05) in genotypic responses to the fungus, the filtrate and the toxin inoculations. Time until phytotoxic symptoms developed was inversely related to the concentrations of CFCF and the toxin. Phytotoxic symptoms were produced 6 days after exposure to 50% CFCF and 48 h after exposure to 1000μg ml^-1 of partially purified phaseolinone. A comparison of photomicrographs of the control and toxintreated root tissues revealed no damage to the control roots and extensive damage to epidermal layers of the treated roots, which was evident 48 h after exposure to 100μg ml^-1 level of phaseolinone. Significant correlations were found between tolerance to the fungus and insensitivity to the culture filtrate (r = 0.89, P = 0.05) and the toxin (r = 0.95, P = 0.001) suggesting the possibility of screening for resistance to M. phaseolina using CFCF or phaseolinone. The genotypic reactions to the CFCF were also correlated with reactions to the toxin (r = 0.90, P = 0.05). Guayule breeding lines‘UC101 and‘P3-1 exhibited the greatest tolerance to the pathogen and insensitivity to the CFCF or the toxin whereas‘Ca16′,‘Cal7′,‘N576′,‘N9-5′, 11605’and‘N6-5’were very susceptible to the pathogen and sensitive to the CFCF or the toxin.     

Production of Host‐selective SV‐toxins by Stemphylium sp. Causing Brown Spot of European Pear in Japan

Recently, a new disease known as ‘brown spot of European pear’ caused by Stemphylium sp. appeared on the leaves, twigs and fruits of the cultivar Le Lectier in Niigata Prefecture, Japan. Because Stemphylium vesicarium (teleomorph: Pleospora allii), which causes a similar disease in Europe, has been shown to produce host‐selective SV‐toxins in culture filtrates (CFs), SV‐toxin production by Stemphylium sp. in Japan was investigated. In pathogenicity tests, the pathogen induced severe necrotic spots on the leaves of the European pear cultivar Le Lectier, slight spots on cultivar La France and slight or no spots on cultivar Bartlett. The Japanese pear cultivar Nijisseiki was not affected by the pathogen. Culture filtrates of the pathogen were tested for phytotoxicity on cultivars by a leaf necrosis assay. The sensitivity of cultivars to the CFs was consistent with the susceptibility of cultivars to the pathogen infection, indicating the presence of host‐selective toxins. The toxins were purified from the CFs according to the procedure reported for SV‐toxin purification in S. vesicarium. The results indicated that Stemphylium sp. in Japan produces the same SV‐toxins as S. vesicarium in Europe.     

Changes in Anatomical Features and Protein Pattern of Sunflower Partially Resistant and Susceptible Lines During Infection By Virulence Factors of Sclerotinia Sclerotiorum

Helianthus annuus L. as an oil seed crop is widely grown throughout the world. One of the most destructive diseases of sunflower is stem rot caused by Sclerotinia sclerotiorum. Oxalic acid is the major virulence factor of this necrotrophic pathogen. It is important to further investigate plant responses to this non-specific toxin. Therefore, in the present study, we compared the patterns of total soluble proteins and xylem morphology of partially resistant and susceptible sunflower lines after treatment with Sclerotinia culture filtrate. The basal stems of both lines were treated with 40 mM oxalic acid (pH 3.7) of fungus culture filtrate and samples were collected at 24, 48 and 72 hours post treatment. In SDS-PAGE protein pattern new protein bands appeared in both lines after treatment. These observations suggest induction of stress-related proteins upon culture filtrate treatment. The identities of these new proteins need to be more clarify in future investigations. The changes in xylem morphology and degree of lignification of both lines was studied by light microscopy and microtome sectioning techniques after treatment with S. sclerotiorum culture filtrate. Anatomical investigations revealed changes in xylem diameter and xylem lignification of treated lines at various time points. More lignin deposition in xylem vessels of partially resistant line has been observed after treatment. In addition, the size of xylem vessels in partially resistant line has been sharply decreased upon pathogen filtrate treatment. The results of this study will help us gain a more complete understanding of resistance mechanisms to this cosmopolitan and devastating pathogen.     

Isolation and Partial Characterization of a Staphylococcal Leukocyte Cytotaxin

A factor which attracts rabbit polymorphonuclear leukocytes both in vitro and in vivo was isolated from the culture filtrate of a strain of Staphylococcus aureus. The activity of the chemotactic factor was independent of fresh serum and it was nondialyzable. Incubation of the factor with heat-inactivated human serum markedly inhibited its chemotactic property. The factor was heat-labile (80 C, 10 min) in the crude culture filtrate but was heat-stable when partially purified.     

Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana

Cullus cultures of elm (Ulmus americana L.) derived from Dutch elm disease susceptible, intermediate-resistant, and resistant genotypes were exposed to the culture filtrates of three patogenic isolates of Ceratocystis ulmi, the causal agent of Dutch elm disease. Callus fresh weights, cell viability, and reactions of stem cuttings were determined after exposure to various concentrations of the filtrates. Calli from the susceptible elm failed to increase in fresh weight and lost viability after exposure to media containing culture filtrate. Calli from the resistant and the intermediate-resistant elms exhibited growth rates and maintained viability similar to controls not exposed to culture filtrate. Stem cuttings of the susceptible elm wilted after exposure to the culture filtrate. The symptoms were similar to wilt seen with the disease. Cuttings from the resistant elm had no disease symptoms whereas, the intermediate elm had some leaf chlorosis. Callus screening may thus be useful for selection of Ulmus germplasm for Dutch elm disease resistance.     

Enterotoxic effects of Aeromonas sobria haemolysin in a rat jejunal perfusion system identified by specific neutralization with a monoclonal antibody.

Investigations into the pathogenesis of Aeromonas diarrhoea have demonstrated that several different cell-free products of motile aeromonads show enterotoxic activity in suckling mouse, rat, and rabbit assay systems. The relative contributions made by separate cytotoxic and cytotonic activities in the mixture produced by in vitro culture remains unresolved. Using a modified rat jejunal perfusion assay, we have studied the effects of A. sobria culture filtrates containing defined levels of haemolytic and cytotoxic activity and immunoreactivity for anti-cholera toxin. This material induced net water, potassium, and sodium loss with a rapid onset (less than 5 min) that was readily differentiated from the effects of purified cholera toxin (greater than 15 min). In filtrates containing up to 128 haemolytic and cytotoxic units of activity, the enterotoxic activity was neutralized by an anti-haemolysin/cytotoxin monoclonal antibody. No specific histological changes could be found in preparations perfused with enterotoxic material for up to 65 min. These findings indicate that the cytotoxic/haemolytic component of A. sobria culture filtrate is the dominant enterotoxic activity.     

Influence of pollen selection for Alternaria helianthi resistance on the progeny performance against leaf blight in sunflower (Helianthus annuus L.)

Seven genotypes of sunflower, including populations and hybrids, showing differential susceptibility to Alternaria leaf and stem blight were crossed to CMS 234A with pollen selection and without pollen selection. The pathogen culture filtrate was used as selective pressure at stylar tissue by applying it 1 h before pollination. Distilled water applied to stigmas and styles served as control. Two sets of seven hybrids, one set from selective and the other from non-selective fertilization, were evaluated for reaction to Alternaria leaf and stem blight during the rainy season under natural epiphytotic conditions. Selection for resistant pollen on the stigmatic surface resulted in a corresponding increase in progeny resistance. The study demonstrates the transmission of the selected trait from the pollen generation to progeny. Further, it was observed that the effect of pollen selection was high in the progenies of moderately resistant parents compared to progenies of highly susceptible parents. The effect of successive pollen selection was studied by backcrossing the progeny derived through selective fertilization to the fertile parent using selective fertilization. Successive pollen selection further improved disease resistance of progeny. However, the improvement was not very great. Hence, repeated cycles of selection are required to achieve a useful level of resistance in the case of sunflower, since resistance is polygenetically controlled. Received: 17 May 1999 / Revision accepted: 13 September 1999     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Induction of systemic resistance in different varieties of Solanum tuberosum by pure and crude elicitor treatment

A 10 kD elicitor protein (infestin) produced by Phytopthora infestans was purified and its efficacy for induction of systemic resistance in resistant and susceptible varieties of Solanum tuberosum was studied. Culture filtrates from P. infestans with and without purified elicitor (infestin) were used as elicitors to understand the effect of purified elicitor (infestin) on development of systemic resistance. Culture filtrate and purified elicitor (infestin) were found to induce hypersensitive reaction on the leaves of resistant varieties, but not on susceptible varieties after 48 h. Culture filtrate devoid of purified elicitor (infestin) did not induce any necrotic spots even on resistant variety. Purified elicitor (infestin) was found to induce glucose oxidase, NADPH oxidase, superoxide dismutase, glutathione reductase, catalase and peroxidase enzymes in resistant S. tuberosum plants, however the induction of these enzymes was low in susceptible varieties. The oxidative enzymes were found to induce earlier than antioxidative enzymes and there was negative correlation between these two groups of enzymes. Levels of salicylic acid, phenylalanine ammonia lyase (PAL), beta-1, 3 glucanase and chitinase activities were also found higher in resistant than in susceptible varieties. It was observed that purified elicitor (infestin) was superior to crude culture filtrate, but was not capable of inducing systemic resistance in susceptible varieties.     

Suppression of phenylalanine ammonia-lyase activity elicited in date palm by Fusarium oxysporum f. sp. albedinis hyphal wall elicitor

The inoculation of the seedling roots of the resistant (Bousthami Noir) and susceptible (Jihel) date palm (Phoenix dactylifera) cultivars by Fusarium oxysporum f. sp. albedinis induced an increase in phenylalanine ammonia-lyase (PAL) activity. The response of the PAL activity in the resistant cultivar was faster and higher than in the susceptible one. However, the elicitation of the seedlings with the hyphal wall elicitor (HWE) of the pathogen induced identical PAL activity in both cultivars. In the resistant cultivar, the the PAL activity elicited with the HWE was not influenced by the addition of the fungal culture filtrate (FCF) whereas it was suppressed in the susceptible cultivar. This FCF suppressor effect was dose-dependent, not influenced by sodium periodate, whereas it was strongly reduced by the heat (121 °C for 45 min) and pronase E. These results show that differential induction of the defence mechanisms in both cultivars was not related to differences in the induction of the PAL activity, but to the suppression of its elicitation in the susceptible cultivar.     

In vitro selection of Coffea arabica callus for resistance to partially purified phytotoxic culture filtrates from Colletotrichum kahawae

Calli derived from hypocotyl explants of a susceptible and resistant genotype of Coffea arabica were evaluated for their response to different concentrations of partially purified culture filtrates (PPCFs) produced by Colletotrichum kahawae which are phytotoxic. The size of calli was measured non-destructively by automated image analysis. Differential responses of calli ranged from complete necrosis or reduced growth in the susceptible genotype (N39) to an absence of necrosis and rapid growth in the resistant genotype (cv. Hybrido de Timor). Subsequently, one selection cycle in the presence of PPCF was devised and applied to calli of nine C. arabica genotypes. Normal plants were regenerated through somatic embryogenesis of callus lines that survived the phytotoxin treatment and in vitro and in vivo testing of these plants against the PPCF showed that increased resistance to the toxin had been obtained. These studies suggest that in vitro selection of calli may be a feasible approach to acquiring germplasm with improved resistance to coffee berry disease.     

Screening for disease resistance in barley cultivars against Bipolaris sorokiniana using callus culture method

Screening for resistant barley genotypes in response to fungal toxin of Bipolaris sorokiniana was assessed on standing barley plants as well as in selected callus lines of the same. For the standing lines tested, those manifesting chlorosis in response to toxin infiltration showed a significantly slower disease progress as compared to the necrotic lines. Also, necrosis in the callus tissues of the susceptible cultivar in MS medium supplemented with different concentrations of the crude toxin was significantly higher than in the callus tissues of the chlorotic lines studied. Similar host response to the toxin in in vitro and field situations open up the possibility of screening barley cultivars for resistance to spot blotch using callus culture as against classical methods of screening in order to increase accuracy and save time and space.     

Isolation and Partial Purification of a Phytotoxin Related to Pathogenic Verticillium species

The low molecular weight metabolites are considered as phytotoxins of pathogenic Verticillum species. The resin, Amberlite XAD-4 was used for isolation of toxin from log-phase culture fluids of the fungus. The toxin was purified further using, gel permeation. The isolated toxin induced chlorosis and necrosis in detached-leaflets of susceptible tomato and potato cultivars similar to natural symptoms. The metabolite is polar, multifunctional, heat stable, cold labile with molecular weight less than 1000. The activity was retained in dried samples of the crude filtrate and kept at room temperature for up to 8 months.