Antimicrobial Activity of Culture Filtrate of Bacillus amyloliquefaciens RC-2 Isolated from Mulberry Leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.     

Purification and Biological Characterization of Host-Specific SV-Toxins from Stemphylium vesicarium Causing Brown Spot of European Pear

ABSTRACT Culture filtrates of a pathogenic isolate (IT37) of Stemphylium vesicarium, causing brown spot of European pear, induced veinal necrosis only on pear leaves susceptible to the pathogen. Two host-specific toxins, SV-toxins I and II, were purified from culture filtrates of IT37 by successively using Amberlite XAD-2 resin adsorption, cellulose thin-layer chromatography, and high-performance liquid chromatography under three different sets of conditions. Susceptible cultivars showed veinal necrosis at a SV-toxin I concentration of 0.01 to 0.1 mug/ml, whereas resistant cultivars were insensitive to the toxin at 1,000 mug/ml. SV-toxins I and II caused a dose-dependent increase in electrolyte loss from susceptible leaf tissues. No increase in electrolyte loss was detected in leaf tissues from resistant cultivars. The results of physiological studies indicated that SV-toxins appear to have an early effect on plasma membranes of susceptible leaves. Spores of a nonpathogenic isolate induced necrotic lesions on susceptible leaves in the presence of a small amount of toxin. SV-toxins were detected in intercellular fluids obtained from diseased leaves after inoculation with the pathogen. The results indicate that SV-toxins I and II produced by S. vesicarium can be characterized as host-specific toxins.     

Biotin Induces Sporulation of Mulberry Anthracnose Fungus, Colletotrichum dematium

Compounds in mulberry leaves inducing sporulation of C. dematium, mulberry anthracnose fungus, were detected. Dissolved or suspended aqueous solutions (1%) of 16 amino acids and 10 vitamins occurring in mulberry leaves were applied individually at the margin of the fungal colony growing on PSA plate. Sporulation was induced only where a biotin solution was applied on the mycelium at a concentration of at least 0.01 ppm. This result suggests that biotin, which occurs in mulberry leaves (ca. 0.6 mg/kg of dried leaves), has a role in inducing sporulation of C. dematium. Received 20 August 1999/ Accepted in revised form 16 November 1999     

Correlation between sensitivity of barley to Pyrenophora teres toxins and susceptibility to the fungus

Detached leaves of 25 barleys, ranging from highly susceptible to highly resistant to Pyrenophora teres f. teres and Pyrenophora teres f. maculata, were tested for their reaction to three phytotoxins isolated from cultures of the fungus: toxin A [ L, L - N -(2-amino-2-carboxyethyl)aspartic acid], toxin C (aspergillomarasmine A) and toxin B (anhydroaspergillomarasmine A). 0.75 m M toxin A caused mainly dark yellow chlorotic symptoms but little necrosis, whereas leaves treated with 0.25 m M toxin C developed distinct necrotic symptoms and zones of light yellow chlorosis. Toxin B is only weakly toxic, and toxin B and control solutions containing aspartic acid in the concentration of 0.75 m M did not cause any symptoms. The best differentiation between the barleys was obtained by scoring chlorosis after 120 h, and the optimal toxin concentrations for this differentiation were 0.75 m M toxin A and 0.25 m M toxin C, respectively. Results with different toxin concentrations inducing distinct variation in symptom expression indicate that the two toxins have different potencies as phytotoxins. The reaction of the barleys to toxins A and C correlated well with their reaction to infection by P. teres f. teres and P. teres f. maculata, suggesting that toxins A and C may be used to select resistant barley lines in the early stages of a breeding programme.     

海南省温郁金炭疽病的病原鉴定

温郁金炭疽病是一种重要的真菌性病害,该病主要危害叶片,初期产生水渍状的淡黄色病斑,后逐渐扩大成圆形、椭圆形或不规则形的灰褐色病斑,上面有黑色小点,病斑外面环绕一层黄色晕圈,严重时多个病斑联合造成叶片卷缩和枯死,2016年我们从海南省的温郁金种植基地采集炭疽病样本,经病原菌的分离纯化、柯赫氏法则验证、病原菌的形态特征观察及多基因谱系分析方法,确定其病原菌为郁金炭疽菌Colletotrichum curcumae(Syd.)E.J.ButlerBisby。     

Myrothecium leaf spot of mulberry caused by Myrothecium verrucaria

A fungus that formed white colonies with greenish black spore masses was isolated from diseased mulberry leaves in Gunma Prefecture, Japan, in 1995, 1999, and 2000. It was pathogenic to mulberry. The fungus was identified with Myrothecium verrucaria. It has been added to the list of pathogens that cause Myrothecium leaf spot of mulberry and is identified for the first time as a plant pathogen in Japan.     

Physiological effects of the hydrophilic phytotoxins produced by <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, the causal agent of black sigatoka in banana plants

Although Mycosphaerella fijiensis, the causal agent of black sigatoka disease of banana, has been known to produce numerous lipophilic host-selective (HSTs) and nonhost selective phytotoxins (non-HSTs), only recently we have reported that the pathogen also produces hydrophilic phytotoxins. Here we examined the effect of light on the toxicity of the hydrophilic phytotoxins and estimated the electrolyte leakage and H₂O₂ and superoxide generation in detached banana leaves to study their mode of action at the cellular level. Nonhost plant species were also tested to determine whether the toxins are HSTs or non-HSTs. Our results suggest that the hydrophilic phytotoxins are non-HSTs, that their phytotoxicity is not light dependent, and that they may act at the plasma membrane by altering permeability through oxidative damage, by inducing ROS production as part of their mechanism of action.     

Isolation of a phytotoxic isocoumarin from Diaporthe eres‐infected Hedera helix (English ivy) and synthesis of its phytotoxic analogs

BACKGROUND

The fungus Diaporthe eres was isolated from a fungal pathogen‐infected leaf of Hedera helix (English ivy) exhibiting necrosis. It is hypothesized that the causative fungus produces phytotoxins as evidenced by necrotic lesions on the leaves.

RESULTS

The fungus was isolated and grown in Czapek Dox broth culture medium and potato dextrose broth culture medium and identified as Diaporthe eres. The ethyl acetate extracts of the culture broths were phytotoxic to lettuce (Lactuca sativa) and bentgrass (Agrostis stolonifera). 3,4‐Dihydro‐8‐hydroxy‐3,5‐dimethylisocoumarin ( 1 ) and tyrosol ( 2 ) were isolated and identified as the phytotoxic constituents. Six analogs of 3,4‐dihydro‐isocoumarin were synthesized and shown to be phytotoxic. The synthesized 3,4‐dihydro‐8‐hydroxy‐3,7‐dimethylisocoumarin and 3,4‐dihydro‐8‐hydroxy‐3,3,7‐trimethylisocoumarin were two‐ to three‐fold more phytotoxic than the naturally occurring 1 in a Lemna paucicostata growth bioassay.

CONCLUSION

Synthesis and herbicidal activities of the several new analogs of 1 are reported for the first time. These promising molecules should be used as templates for synthesis and testing of more analogs. © 2017 Society of Chemical Industry     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

Occurrence and development of Colletotrichum acutatum on symptomless blueberry bushes

The anthracnose fungus Colletotrichum acutatum was detected in symptomless blueberry bushes ( Vaccinium spp.) in a Japanese blueberry field. Naturally diseased bushes and their apparently healthy neighbours were selected, and C. acutatum was isolated from the symptomless tissues of each bush from February 2000 to January 2002. Analysis of the diseased bushes during the dormant period revealed that the fungus was able to survive on symptomless tissues, such as shoot bark and bud scales. Furthermore, C. acutatum was consistently isolated from symptomless leaves and shoots of several surrounding symptomless bushes. Arbitrarily primed PCR (ap-PCR) analyses of the fungal isolates obtained from the diseased and symptomless bushes revealed that most C. acutatum isolates were genotypically identical, regardless of their origins. Inoculation tests using leaves of various blueberry cultivars suggested that the presence or absence of symptoms on each bush can not always be explained by differences in cultivar susceptibility, and other factors may be associated with the appearance of symptoms.     

Cultivar Resistance to Anthracnose Disease of Cowpea (Vigna unguiculata (L.) Walp.) caused by Colletotrichum destructivum O'Gara

The infection process of Colletotrichum destructivum, a hemibiotrophic anthracnose fungus, was studied by light microscopy in two cowpea (Vigna unguiculata) cultivars which differ in disease reaction type. Large, multilobed, intracellular infection vesicles, followed by necrotrophic, radiating, secondary hyphae were produced in tissues of the susceptible cv. IT82E-60. In the resistant cv. TVx3236, both the production of appressoria and their melanisation were impaired, resulting in reduced penetration. Where penetration occurred, the initially-infected epidermal cells underwent a hypersensitive response, restricting the growth of multilobed vesicles and thereby blocking the destructive necrotrophic phase of disease development. The phytoalexins kievitone and phaseollidin accumulated earlier and more rapidly in stem tissues of the resistant cultivar, associated with the appearance of delimited, necrotic spots on inoculated surfaces. In contrast, delayed and slower accumulation of these compounds occurred in the compatible interaction, together with the development of typical spreading, water-soaked, anthracnose lesions.     

Stomatal penetration of cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing latent anthracnose

The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea ( Vigna unguiculata ) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting > 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C . gloeosporioides from Aeschynomene virginica, Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum .     

Fusaproliferin effects on the photosystem in the cells of maize seedling leaves

The possible role of the fusariotoxin, fusaproliferin in plant pathology was investigated with respect to cell membrane potential. Electron microscopy was used to study both the early effect of fusaproliferin on the host’s plasma membrane and ultrastructure responses in the cells of maize leaves. The seedlings of resistant (Lucia) and susceptible (Pavla) to the fusaproliferin maize cultivars were grown in the presence of fusaproliferin at different concentrations, namely 5 and 35 μg ml⁻¹, respectively, and electrophysiological measurements were compared with those obtained using two different toxic compounds, namely fusicoccin and 3-3(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU). It was observed that only the higher concentration of fusaproliferin induced the onset of visible symptoms on the leaves. Comparing the effect of fusaproliferin to that of fusicoccin and DCMU at the higher toxin concentration, it was observed that functional differences in membrane potential induced severe damage to the mesophyll and outer chloroplast membrane; the extent of changes in electrophysiology and ultrastructure disturbances depended on the toxin concentration and was greater in the susceptible cv. Pavla. Results indicated that fusaproliferin could be involved in Fusarium pathogenesis either as a virulence factor or by enhancing the activity of other toxins that might be concomitantly present in infected plants.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

The effects of rust and anthracnose on the photosynthetic competence of diseased bean leaves

ABSTRACT The effects of rust (caused by Uromyces appendiculatus) and anthracnose (caused by Colletotrichum lindemuthianum) and their interaction on the photosynthetic rates of healthy and diseased bean (Phaseolus vulgaris) leaves were determined by gas-exchange analysis, in plants with each disease, grown under controlled conditions. The equation P(x)/P(0) = (1 - x)() was used to relate relative photosynthetic rate (P(x)/P(0)) to proportional disease severity (x), where beta represents the ratio between virtual and visual lesion. The beta values obtained for rust were near one, indicating that the effect of the pathogen on the remaining green leaf area was minimal. The high values of beta obtained for anthracnose (8.46 and 12.18) indicated that the photosynthesis in the green area beyond the necrotic symptoms of anthracnose was severely impaired. The impact of anthracnose on bean leaf photosynthesis should be considered in assessments of the proportion of healthy tissue in diseased leaves. The accurate assessment of the healthy portion of the leaf could improve the use of concepts such as healthy leaf area duration and healthy leaf area absorption, which are valuable predictors of crop yield. The equation used to analyze the interaction between rust and anthracnose on the same leaf was P(z) = P(0) (1 - x)(x) x (1 - y)(y), where P(z) is the relative photosynthetic rate of any given leaf, P(0) is the maximum relative photosynthetic rate, x is anthracnose severity, y is rust severity, betax is the beta value for anthracnose in the presence of rust, and betay is the beta value for rust in the presence of anthracnose. From the resulting response surface, no interaction of the two diseases was observed. Dark respiration rate increased on diseased leaves compared with control leaves. The remaining green leaf area of leaves with both diseases was not a good source to estimate net photosynthetic rate because the effect of anthracnose extended far beyond the visual lesions, whereas the effect of rust on photosynthesis was essentially limited to the pustule plus halo.     

Suppression of rice blast using freeze-killed mycelia of biocontrol fungal candidate MKP5111B

Several applications of the suppressive fungus MKP5111B, isolated from the phylloplane of rice plants, were tested in an effort to control rice blast disease. Three treatments with MKP5111B [living (Std), killed with liquid nitrogen (FR), and autoclaved (AC)] were either sprayed onto rice seedlings or mixed into seed-sown soil. Three weeks after spraying and 4 weeks after the soil application, we introduced Magnaporthe oryzae, the causal agent of rice blast, into our systems. The Std and FR treatments suppressed rice blast, but the AC treatment proved ineffective. Although a suppressive effect was seen on new leaves, no mycelium of MKP5111B was seen. The fungus thus may have induced a systemic resistance in the rice plants. A substance from MKP5111B, such as elicitor molecule(s) are likely responsible for the induced resistance.     

Sensitivity among species of Solanaceae to AAL toxins produced by Alternaria alternata f.sp. lycopersici

The necrotrophic fungus Alternaria alternata f.sp. lycopersici produces AAL toxins that cause necrosis in tomato tissues with high specificity. Resistance or susceptibility of tomato to the fungus and insensitivity or sensitivity to AAL toxins are determined by a single locus, Asc . In order to investigate further the specificity of the host–fungus interaction, 200 species of Solanaceae were tested for their sensitivity to AAL toxins T_A and T_B. Twenty-five species were found to be sensitive to AAL toxins at a concentration of (0·2  µ m ) used for distinguishing sensitive and insensitive tomato plants. Three species were as sensitive as the sensitive tomato line, indicating that AAL toxins effectively act on a broader range of plant species within the Solanaceae.     

Structural Modifications and Programmed Cell Death of Chili Pepper Fruit Related to Resistance Responses to Colletotrichum gloeosporioides Infection

ABSTRACT Postharvest (detached) and in planta (attached) fruits of pepper plants, Capsicum annuum cv. Jejujaerae (susceptible) and Capsicum baccatum cv. PBC80 (resistant), inoculated with the anthracnose pathogen Colletotrichum gloeosporioides were examined using light, confocal laser scanning, and electron microscopy to compare the cytological differences between the compatible and incompatible interactions. In nonwound inoculation of postharvest pepper fruit, resistant pepper tissues showed a significant increase in the thickness of the cuticle layer compared with that of the susceptible and noninoculated fruit. Cytological features of programmed cell death (PCD) were observed in the resistant pepper fruit with postharvest inoculation, and these were characterized by positive responses to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The oligonucleosomal fragments of DNA were confirmed electrophoretically as DNA laddering. The PCD-positive responses occurred around the inoculation sites early in in planta wound inoculation in the resistant pepper. Nuclear modifications and structural changes of hypersensitivity were also observed in the resistant fruit, including separation of the plasma membrane from the cell wall, dilation of the endoplasmic reticulum, accumulation of electron-dense inclusions in vacuoles, and cytoplasmic vacuolization accompanying fragmentation of the cytoplasm. These structural changes may also implicate PCD-like host responses. In addition, in planta wound inoculation resulted in cell enlargement and cell division during the later stages of infection to form a periderm-like boundary layer around the inoculation site.