Development of Colletotrichum gloeosporioides Restriction Enzyme-Mediated Integration Mutants as Biocontrol Agents Against Anthracnose Disease in Avocado Fruits

ABSTRACT Reduced-pathogenicity mutants of the avocado fruit pathogen Colletotrichum gloeosporioides isolate Cg-14 (teleomorph: Glomerella cingulata) were generated by insertional mutagenesis by restriction enzyme-mediated integration (REMI) transformation. Following seven transformations, 3,500 hygromycin-resistant isolates were subjected to a virulence assay by inoculation on mesocarp and pericarp of cv. Fuerte avocado fruits. Fourteen isolates showed a reduced degree of virulence relative compared with wild-type Cg-14. Two isolates, Cg-M-142 and Cg-M-1150, were further characterized. Cg-M-142 produced appressoria on avocado pericarp similar to Cg-14, but caused reduced symptom development on the fruit's pericarp and mesocarp. Isolate Cg-M-1150 did not produce appressoria; it caused much reduced maceration on the mesocarp and no symptoms on the pericarp. Southern blot analysis of Cg-M-142 and Cg-M-1150 showed REMI at different XbaI sites of the fungal genome. Pre-inoculation of avocado fruit with Cg-M-142 delayed symptom development by the wild-type isolate. Induced resistance was accompanied by an increase in the levels of preformed antifungal diene, from 760 to 1,200 mug/g fresh weight 9 days after inoculation, whereas pre-inoculation with Cg-M-1150 did not affect the level of antifungal diene, nor did it delay the appearance of decay symptoms. The results presented here show that reduced-pathogenicity isolates can be used for the biological control of anthracnose caused by C. gloeosporioides attack.     

The relationship between antifungal diene levels and fungal inhibition during quiescent infection of unripe avocado fruits by Colletotrichum gloeosporioides

Quantitative changes in the antifungal compound, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12.15-diene, in freshly harvested avocado fruits during the initial stages of fungal development were investigated to determine the possible involvement of the compound in quiescent infections of Colletotrichum gloeosporioides. The concentration of the antifungal compound in the peel decreased to subfungitoxic concentrations 16 h after harvest. Fifty-six hours later the antifungal diene had increased to c . 3800 μg/g fresh weight. At this stage, germinated appressoria had penetrated the cuticle to the epidermal cells but no fungal development was observed until 7 days later when the concentration of the diene had decreased to 100-110μg/g fresh weight. Following a dip treatment at 55°C for 5 or 10 min, the antifungal diene concentration decreased as in the controls, but it remained at subfungitoxic concentrations for a longer period enabling fungal development and early symptom expression.
The concentration of the diene in the flesh of freshly harvested fruit decreased to 120 μg/g fresh weight 24 h after harvest. Inoculation of peeled fruits with spores of C. gloeosporioides showed germination without appressoria formation and symptom expression occurred 24-48 h later. Symptom expression was delayed if fruits were inoculated after coating the flesh with epicuticular wax extracts or if the flesh was inoculated 3 days after harvest when the antifungal diene had regained a fungitoxic concentration. Disease symptoms were expressed in soft fruits containing subfungitoxic concentrations of the diene.
We conclude that the diene in unripe avocado fruits inhibits fungal development of germinated appressoria or conidia. The quiescent structure of C. gloeosporioides in unripe avocado fruit is a subcuticular hypha.     

Antifungal activity, acid and sugar content in the wood apple (Limonia acidissima) and their relation to fungal development

Unripe wood apple fruit is generally free from visible fungal growth before and at harvest but a succession of fungi appears on the fruit shell, and sometimes in the pulp, during ripening. A TLC- Cladosporium bioassay of the chloroform extract taken from unripe fruit shell demonstrated three inhibition areas. Similar extracts from stem-bark and root-bark produced these three, and one additional, inhibition areas. The four compounds responsible for inhibition were identified as psoralene, xanthotoxin, 2,6-dimethoxybenzoquinone and osthenol.
Concentrations of the three antifungal compounds on unripe fruit shell increased during the first 4 days after harvest and then declined. They remained much below those required to inhibit the development of three fungi on TLC plates. Titratable acidity of the unripe fruit pulp was high but decreased by about 50% during ripening. Levels of reducing sugars were very low in the unripe fruit pulp but increased by about five times during ripening. Levels also increased in the fruit shell and its washings. The possible role of these factors in restricting fungal growth in unripe fruits is discussed.     

Phylogenetic and morphological identification of Colletotrichum boninense: a novel causal agent of anthracnose in avocado

In recent years, anthracnose has become a significant disease affecting avocado fruit in the state of Michoacan, Mexico, where it significantly reduces fruit quality and commercial yield. Anthracnose has been assumed to involve Colletotrichum gloeosporioides and C. acutatum as causal agents. However, because of the increasing incidence of anthracnose, a more precise identification of the Colletotrichum spp. involved in this disease has become desirable. During the years 2004–2007, avocado fruits of different sizes exhibiting brown‐black and reddish spots on the pericarp and soft rot in the mesocarp, were gathered from orchards in nine counties. Fungal isolates were cultured on potato dextrose agar, and among these, 31 were selected for molecular, morphological and pathogenicity analyses. The molecular approaches used sequence typing of the internal transcribed spacer region and the partial nuclear large ribosomal subunit, allowing the unequivocal identification of C. gloeosporioides (71%), C. acutatum (16%) and C. boninense (13%). This last species has not been previously reported as being associated with anthracnose symptoms in avocado fruits anywhere in the world. Various morphological characteristics such as the size and shape of conidia were determined, as well as the conidial mass colour. Pathogenicity tests performed with all three species were conducted by inoculating healthy fruits. In each case, identical symptoms developed within 3 days of inoculation. Knowledge of the Colletotrichum populations in the Michoacan state, including the newly encountered avocado pathogen C. boninense, will facilitate further studies addressing the relationships between these Colletotrichum spp. and their avocado host.     

The effect of hot-water treatment on the levels of antifungal diene and quiescence of Colletotrichum gloeosporioides in avocado fruits

Quantitative changes in the antifungal compound 1-acetoxy-2-hydroxy-4-Oxo-heneicosa-12, 15-diene in harvested avocado fruits, and the development of symptoms caused by Colletotrichum gloeosporioides , were investigated following treatment with hot water at 55°C for 10 min. The concentration of the compound in the peel and flesh was 2000 and 2600 μg/g fresh weight, respectively, at the time of harvest, but decreased rapidly during the first 24 h. Levels of the diene had substantially recovered after 50 h. However antifungal diene levels in the peel of hot-water-treated fruit did not recover until 98 h. The levels in the flesh were unaffected by the treatment. Following inoculation, hot-water-treated fruits developed clear symptoms after 2 days, whereas untreated fruits showed only minor symptoms after 6 days. If inoculation was delayed by 24, 48 or 72 h after treatment, then symptoms on treated fruits did not develop until the sixth day as observed for untreated fruits. The correlation between the two systems suggests that quiescence is probably maintained by the level of antifungal diene present in the peel at the time of fungal penetration and the formation of a subcuticular hypha.     

Penicillium digitatum Suppresses Production of Hydrogen Peroxide in Host Tissue During Infection of Citrus Fruit

ABSTRACT During the infection of citrus fruit by Penicillium digitatum there is little evidence of a host defense response. This suggests that P. digitatum has the ability to suppress host defenses. The current study demonstrates that P. digitatum suppresses a defense-related hydrogen peroxide (H(2)O(2)) burst in host tissue. In contrast, the nonhost pathogen, Penicillium expansum, triggers production of a significant amount of H(2)O(2) in citrus fruit exocarp. Using laser scanning confocal microscopy, we demonstrated that P. digitatum suppressed an elevation in H(2)O(2) up to 42 h after inoculation. Nevertheless, H(2)O(2) levels around wounds inoculated with P. expansum increased by 63-fold above the control. P. digitatum continued to suppress H(2)O(2) production in citrus fruit exocarp up to 66 h postinoculation and H(2)O(2) levels were actually threefold below that of noninoculated controls. In contrast, the H(2)O(2) level was still about 11-fold above the control value in wound sites inoculated with P. expansum. Studies on the effect of organic acids (as pH modulators) on the response of citrus fruit to compatible and noncompatible pathogens indicated that pathogenicity was enhanced only when host-tissue acidification was accompanied by the suppression of H(2)O(2). Additionally, pathogenicity of both P. digitatum and P. expansum on citrus fruit was significantly enhanced by the H(2)O(2)-scavenging enzyme catalase. Based on our study and previous reports regarding the potential involvement of citric acid and catalase in green mold pathogenesis, we suggest that these compounds are strongly associated with the virulence of P. digitatum.     

Latency- and Defense-Related Ultrastructural Characteristics of Apple Fruit Tissues Infected with Botryosphaeria dothidea

ABSTRACT Apple fruit tissues infected with Botryosphaeria dothidea were examined by transmission electron microscopy using susceptible cv. Fuji and resistant cv. Jonathan. Immature (green) and mature (red) fruits of cv. Fuji with restricted or expanding lesions were also examined to reveal subcellular characteristics related with latent and restricted disease development. In infected susceptible mature fruits, cytoplasmic degeneration and organelle disruption commonly occurred, accompanying cell wall dissolution around invading hyphae. Cell wall dissolution around invading hyphae in subepidermis was rare in immature, red halo-symptomed cv. Fuji and resistant cv. Jonathan fruits. In infected immature fruits of cv. Fuji, presumably at the latent state of disease development, cellular degeneration was less severe, and invading hyphae contained prominent microbody-lipid globule complexes or the deposition of thin electron-dense outer layer around cell wall of intercellular hyphae. Both mature fruits with red halos and resistant apple fruits formed cell wall protuberances at the outside of cell walls. In addition, electron-dense extramural layers were formed in the resistant apple fruits. Aberrant hyphal structures such as intrahyphal hyphae were found only in resistant fruit tissues, indicating the physiologically altered fungal growth. These ultrastructural changes of host tissues and fungal hyphae may reflect the pathogenesis of apple white rot under varying conditions of apple fruits.     

内生放线菌A-1对苹果果实轮纹病的防效及防御性酶活性的影响

为揭示内生放线菌A-1对苹果果实轮纹病的防效及防病机制,采用平板法和喷雾处理刺伤接种法,测定了其对苹果轮纹病菌的抑制作用及果实内防御性酶活性的影响。结果表明：菌株A-1能显著抑制轮纹病菌菌丝生长,10⁴ CFU/mL菌悬液的抑制率达90%以上;喷施10⁷ CFU/mL A-1菌悬液后,间隔12 h以上接种的各处理,3 d和7 d时防效分别为91.79%~95.67%和77.41%~94.00%,均与对照农药苯醚甲环唑相当。喷施10⁷ CFU/mL A-1菌悬液后接种或不接种轮纹病菌的处理,果实内过氧化物酶、过氧化氢酶、多酚氧化酶和苯丙氨酸解氨酶活性显著升高,且均高于只接种轮纹病菌的处理;喷施A-1菌悬液后接种病原菌的处理酶活性增加最显著,其峰值是对照的2.30~11.00倍。表明内生放线菌A-1可通过产生拮抗物质、提高寄主防御性酶活性等机制实现对苹果轮纹病的有效防治。     

Acidified peel of litchi fruits selects for postharvestPenicillium decay

Litchi fruits are fumigated after harvest with sulfur dioxide (SO₂) to prevent their rapid browning. SO₂ blocks enzymatic activity but bleaches the fruits and, if this process is followed by dipping the fruit in dilute hydrochloric acid, the appealing red color is regained. Hot water brushing (HWB) is among the alternative methods that were developed to replace the use of SO₂. HWB reduced fungal population size on the surface of the fruit peel after treatment but did not eliminate fruit infection after storage. Whereas untreated fruits were infected with a variety of fungal species,Penicillium sp. was the only fungus that developed on the pericarp after storage in fruits that had been dipped in 1.5M HCl. Fruit treated by HWB followed by handling and storage under sterile conditions suffered greater decay than fruit stored under non-sterile conditions but with more ventilation. APenicillium sp. isolated from litchi grew well in liquid medium acidified to the pH range reported for SO₂ and HCl-treated litchi fruits. Morphological analysis identified fungal isolates asP. aurantiogriseum. Internal transcribed spacer sequence analysis of five isolates suggested a sequence similarity toP. commune. Our data support the hypothesis that dipping litchi fruit in hydrochloric acid eliminates infection by common opportunistic fungi and selects forPenicillium species that tolerate low pH. http://www.phytoparasitica.org posting April 30, 2004.     

4种化学物质对南瓜抗白粉菌产生过敏性反应影响的研究

研究了4种化学物质对南瓜抗白粉病产生过敏性反应的影响,结果表明H2O2+接菌处理可使南瓜叶片中过敏性细胞数量增加,且随着浓度的升高过敏性细胞数量逐渐增多,经20mmol/L H2O2 + 接菌处理的南瓜叶片,在96h时产生的过敏性细胞比对照多81个/cm2; 而活性氧清除剂过氧化氢酶、抗坏血酸和谷胱甘肽3种化学物质处理+接菌南瓜叶片后,叶片中过敏性细胞的数量显著减少,且浓度越高,过敏性细胞数量越少,2000U/mL过氧化氢酶+接菌、20mmol/L抗坏血酸+接菌和40mg/L谷胱甘肽 + 接菌处理南瓜叶片,96h时过敏性细胞数量分别比对照 + 接菌少79、95和90个/cm2; 接菌10d后,20mmol/L H2O2 + 接菌处理的南瓜叶片的病情指数比对照 + 接菌低14.1,2000U/mL过氧化氢酶 + 接菌、20mmol/L抗坏血酸 + 接菌和40mg/L谷胱甘肽 + 接菌处理的南瓜叶片的病情指数分别比对照高7.47、4.95和6.83。     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

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.     

Management and cross-infectivity potential of Colletotrichum acutatum causing anthracnose on bell pepper in Florida

Early anthracnose caused by Colletotrichum acutatum has become an increasingly serious disease on green, unripe bell pepper fruit in Florida. This contrasts with earlier reports of anthracnose occurring on bell pepper primarily as a ripe-rot disease of mature, colored pepper fruit caused by Colletotrichum gloeosporioides. Management of anthracnose on green bell pepper fruit using fungicides and a commercial inducer of systemic acquired resistance, acibenzolar-S-methyl (ASM), was evaluated during three seasons. In two of the three trials, all the fungicides tested including azoxystrobin, fludioxonil + cyprodinil, mancozeb, famoxadone + cymoxanil, copper hydroxide, and ASM significantly increased the number of marketable fruit compared with control plants. These trials identified fungicides that could contribute to a successful pest management program on pepper for controlling anthracnose caused by C. acutatum. The cross-infectivity potential of C. acutatum was investigated on tomato and strawberry by in vitro and field inoculation. Anthracnose lesions formed readily on wound-inoculated detached fruits of all hosts in in vitro assays. Under field conditions, after inoculation, anthracnose lesions occurred on pepper fruit but no lesions of anthracnose were found on either ripe or unripe tomato or strawberry fruit in adjacent plots.     

Field evaluation of systemic imidacloprid for the management of avocado thrips and avocado lace bug in California avocado groves

BACKGROUND: The efficacy of systemic applications of imidacloprid for the management of avocado thrips and avocado lace bug was determined in field trials. Following insecticide treatment by chemigation, leaves of appropriate age for each insect were sampled over a 6 month period and used for bioassays. Imidacloprid residues were measured by ELISA in leaves used for bioassays to determine concentrations of insecticide that were toxic to both pests. RESULTS: The uptake of imidacloprid into treated trees was extremely slow, peaking in the current year's leaf flush at only 8 ng cm^?2 leaf tissue after 15 weeks. Avocado thrips mortality in bioassays with young flush leaves, the preferred feeding substrate for this insect, was minimal, indicating that imidacloprid concentrations were below threshold levels needed for effective control. Residues present in older leaves, which are preferred by the avocado lace bug, were higher than in young flush leaves, and provided good control of this pest. Probit analysis of bioassay data showed that the avocado lace bug (LC₅₀ = 6.1 ng imidacloprid cm^?2 leaf tissue) was more susceptible to imidacloprid than the avocado thrips (LC₅₀ = 73 ng imidacloprid cm^?2 leaf tissue). CONCLUSIONS: In spite of the slow uptake of imidacloprid into avocado trees, the levels of imidacloprid would be sufficient to control avocado lace bug infestations. In contrast, the slow uptake would be problematic for avocado thrips control because inadequate levels of insecticide accumulate in new flush foliage and would allow avocado thrips populations to build to levels that would subsequently damage developing avocado fruit. Copyright © 2010 Society of Chemical Industry     

In vitro and in vivo toxicity of nano chitosan against Curvularia lunata,the causal microorganism of fruit rot and blight,a new disease of olive (O. europaea L.)

During 2019, fruit blight and rot symptoms were observed on olive (O. europaea L.) fruits on trees grown in the Experimental Farm, Faculty of Agriculture, Sohag University, Egypt. Fungal isolates recovered from symptomatic fruits were identified as Curvularia lunata (Walker) Boedijn (two isolates) and A. alternata (Fr.) Keissl. (one isolate). Koch’s postulates were fulfilled by a pathogenicity test conducted in vitro on olive fruits wounded before inoculation with fungal isolates and incubation at 25?±?0.2 °C in a moist chamber for a week. During incubation, we observed the development of blight and rot symptoms on fruits inoculated with both isolates of C. lunata, similar to the natural symptoms described. Conversely, A. alternata was nonpathogenic to olive fruits. PCR amplification using the specific P1 and P2 primers to C. lunata based on the Clg2p Ras protein gene sequences resulted in approx. 870 base pairs for all DNA of C. lunata analyzed, confirming the identification of C. lunata. In vitro, both chitosan nano and non-nano scale effectively inhibited mycelial growth by reducing linear mycelium and biomass and sporulation of C. lunata. In vivo, chitosan nanoscale at 2.0 mg mL^?1 greatly reduced the infection and the lesion diameter of C. lunata inoculated fruits after a week and effectively induced defense-related enzyme activity of PO, PPO, and PAL. This report is the first recording of fruit blight and rots on olive caused by C. lunata, as a new disease. Also, we report the in vitro and vivo toxicity of nanoparticles of chitosan as a natural elicitor, effectively inducing defense-related enzymes against C. lunata.

     

Stress on avocado fruits regulates Δ9-stearoyl ACP desaturase expression,fatty acid composition,antifungal diene level and resistance to Colletotrichum gloeosporioides attack

The expression of the avocado homologue avfad9 encoding Δ⁹-stearoyl-ACP desaturase was enhanced by multiple stimuli: inoculation with Colletotrichum gloeosporioides, exposure to ethylene or CO₂, low temperature (4 °C) and fruit wounding. This enhanced expression was correlated with an increase in the preformed antifungal (Z, Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene. Treatments of fruits with ethylene that enhanced the up-regulation of avfad9 also increased the concentration of 18:2 fatty acid and the incorporation of ¹⁴C-linoleate into the antifungal diene. Fruits with enhanced Δ⁹stearoyl-ACP desaturase expression were more resistant to C. gloeosporioides. It is suggested that the enhanced Δ⁹stearoyl-ACP desaturase expression is involved in elevation of unsaturated 18:2. It is also concluded that similar treatments enhance the incorporation of labeled 18:2 into the antifungal diene and elicit the concurrent enhanced resistance to fungal attack.     

黄瓜-酸黄瓜渐渗系霜霉病抗性及感病前后几种酶活性的变化

 Using resistant and susceptible cultivars as controls, the resistant evaluation to Pseudoperonospora cubensis was carried out in 12 introgression lines from wide cross between cucumber(Cucumis sativus L.) and sour cucumber(Cucumis hystrix Chakr.). Three enzyme activities including POD, SOD and PAL were analyzed before and 7 d after inoculation, and the POD isozyme was detected by polyacrylamide electrophoresis. The inoculation results showed that, of the 12 accessions, 3 were identified as high resistant, 5 were moderately resistant and 4 were moderately susceptible to downy mildew. The enzyme activities of POD, SOD and PAL were greatly increased in resistant accessions after inoculation. PAL enzyme activities showed close correlation with disease rating before or after inoculation, which implicated that PAL enzyme activity might be used to estimate the resistance to downy mildew. POD isozyme electrophoresis showed that the number and intensity for the bands of resistant lines were significantly increased more than those of susceptible lines after inoculation.