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.     

Resistance of Gibberellin-Treated Persimmon Fruit to Alternaria alternata Arises from the Reduced Ability of the Fungus to Produce Endo-1,4-beta-Glucanase

ABSTRACT Black-spot symptoms, caused by Alternaria alternata, developed in persimmon fruits during prolonged storage at -1 degrees C. A preharvest treatment with gibberellic acid (GA(3)) extended the storage life of the fruit by delaying both black-spot development and fruit softening. Conversely, treatment of persimmon fruits with paclobutrazol (PBZ), an inhibitor of gibberellin (GA) synthesis, enhanced black-spot development and fruit softening during storage. Production of endo-1,4-beta-glucanase (EC 3.2.1.4, EG) by A. alternata in culture and in the presence of cell walls from PBZ-treated fruits as the carbon source, was enhanced by 150% over production in the presence of cell walls from control fruits, whereas endoglucanase (EG) production in the presence of cell walls from GA(3)-treated fruits was reduced by 49% relative to controls. To determine the importance of EG in symptom development, A. alternata EG was purified from a culture-inducing medium. It had a molecular mass of 41 kDa, its optimal pH and temperature for activity were 5.5 and 47 degrees C, respectively, and the pI was 4.3. Its K(m) and V(max) were 0.43 mg ml(-1) and 18 mumol reducing groups minute per milligrams of protein, respectively. The internal sequence of a 21-mer amino acid peptide from the purified EG showed 62% similarity and 38% identity to the EG-1 of Trichoderma reesei and of T. longibrachiatum. Purified EG induced black-spot symptoms on the fruit, similar to those caused by A. alternata, whereas boiled enzyme caused only pricking signs. Our results suggest that the black-spot symptoms caused by A. alternata, in persimmon, are related to the ability of the fungus to produce EG in developing lesions.     

Early Events During Quiescent Infection Development by Colletotrichum gloeosporioides in Unripe Avocado Fruits

Inoculation of avocado pericarp tissue with Colletotrichum gloeospori-oides and treatment of avocado cell cultures with the cell wall elicitor of C. gloeosporioidesboth increased the production of reactive oxygen species (ROS). However, whereas the production of ROS could be detected within minutes in avocado cell suspensions, it was detected only after 2 h following inoculation of pericarp tissue. Protein kinase inhibitors such as K-252a and staurosporine and the phosphatase inhibitor microcystin-LR inhibited the release of H(2)O(2) from avocado cell suspensions. When 1 mM H(2)O(2) was exogenously applied to pericarp tissue, it enhanced ROS, phenyl-alanine ammonia lyase (PAL) activity, and epicatechin levels. But, when H(2)O(2) treatment was applied following staurosporine treatment, PAL activity was no longer induced. The uninduced ROS production in pericarp tissue of freshly harvested, unripe, resistant fruit was twice as high as in ripe, susceptible fruit. Challenge inoculation of resistant fruit further increased the ROS level; however, this increase did not occur in susceptible fruits. The current findings are consistent with the hypothesis that production of ROS is induced by fungal infection of unripe fruits and, consequently, may modulate resistance, resulting in the inhibition of fungal development and quiescence.     

Acetylsalicylic acid (ASA) suppressed Fusarium rot development and neosolaniol (NEO) accumulation by activating phenylpropane metabolism in muskmelon fruit

European Journal of Plant Pathology - Fusarium rot of muskmelon fruit, caused by Fusarium sulphureum, is a typical postharvest decay that not only seriously influences fruit quality but also leads...     

Is the accumulation of a systemic fungicide at the infection site related to its eradicative action?

Restricted areas of Avena sativa leaves were infected with Puccinia coronata. The systemic fungicide [³H]triarimol was later applied to the roots of these plants. The fungicide did accumulate at the infection sites when applied 14 days after inoculation, but not when applied 4 or 9 days after inoculation. It is concluded that the accumulation of the fungicide at the infection zone is a result of enhanced evaporation from leaf tissues (probably) because the epidermis was broken after pustule formation.     

Improved control of moldy-core decay (<Emphasis Type="Italic">Alternaria alternata</Emphasis>) in Red Delicious apple fruit by mixtures of DMI fungicides and captan

The sterol biosynthesis inhibitors bromuconazole and difenoconazole and tank mixes of each fungicide with captan were applied to apples and evaluated as controls for moldy-core and fruit decay caused by Alternaria alternata. Effectiveness of a mixture of bromuconazole and captan in controlling colonization by the fungus was also evaluated. Decay formation by A. alternata on mature detached fruits was partially inhibited by bromuconazole at 0.5 μg ml⁻¹ and was completely inhibited at 50 μg ml⁻¹; it was significantly affected by either bromoconazole at 5 μg ml⁻¹ or captan at 1,250 μg ml⁻¹, and was completely inhibited by their mixture. In general, three foliar applications of bromuconazole or difenoconazole in the field, during the bloom period, reduced the numbers of infected fruits by 40–60% compared with untreated control trees. However, tank mixes of either fungicide with captan improved control of moldy-core in fruits at harvest. Tank mixtures of bromuconazole and captan also significantly reduced the percentage of fruits colonized by A. alternata when sampled at various days after full bloom. Artificial inoculations in the orchard at full bloom did not change the inhibitory effects of the tank mixtures. Large-scale demonstration trials in commercial orchards supported these findings. The inhibitory effects of tank mixes on decay development in detached fruits, and on moldy-core in the field indicate that a control programme based on mixtures of either bromuconazole or difenoconazole with captan during the bloom period can effectively reduce moldy-core on Red Delicious apples.     

Sensitivity of Red Delicious Apple Fruit at Various Phenologic Stages to Infection by Alternaria alternata and Moldy-core Control

The fungus Alternaria alternata, is considered to be the predominant fungus involved in moldy-core of Red Delicious strains of apple. In this paper, we report on the sensitivity of various phenologic stages to infection by A. alternata, and on the efficacy of various fungicides in controlling moldy-core disease in apple orchards. Artificial inoculations conducted in the orchard during 1999 and 2000 seasons revealed that the beginning of bloom (10–30%) and full bloom were the most susceptible developmental stages for infection. Natural infection with A. alternata in fruits was relatively high, reaching 44% and 46% of the fruits on control non-treated trees in 1999 and 2000. Four foliar applications of polyoxin B, difenoconazole and azoxystrobin, starting from the beginning of bloom until fruit set, reduced the number of infected fruits by 54–70%, 61–70% and 50–65%, respectively, compared with non-treated trees. Four or eight foliar applications of each fungicide provided similar levels of control. There were no significant differences between two, four or six foliar applications of difenoconazole, neither between two or four applications of polyoxin B. Adding CaCl₂, as a tank mixture with difenoconazole at full rate, did not improve efficacy. Alternaria was recovered from the inner part of the core region of 71–88% of the fruits of the non-treated control, but was recovered less frequently from the outside part of the core region. Fruits of difenoconazole and polyoxin B treated trees were less colonized with A. alternata at both the inner and outside parts of the core region, as compared with controls. Results indicate that a control programme based on spray applications of difenoconazole or polyoxin B, during bloom period, can effectively reduce Alternaria on Red Delicious.