Sensitivity of Colletotrichum gloeosporioides Isolates from Diseased Avocado Fruits to Selected Fungicides in Kenya

Colletotrichum gloeosporioides is a serious postharvest pathogen of avocado fruits worldwide. Kenya lacks any registered fungicides for the management of the disease. Nevertheless, farmers commonly use commercially available fungicides such as Bayleton 25WP (Triadimefon 250 g/Kg), Milraz 76WP (Propineb 70% and Cymoxanil 6%), and Copper oxychloride 500WP for disease management. The efficacy of these fungicides against C. gloeosporioides is not known. The purpose of this study was therefore to test the inhibitory effect of these fungicides against 46 C. gloeosporioides isolates from avocado fruits collected from varieties grown at different agroecological zones in Murang’a County, a popular avocado-growing region in Kenya. Mycelial growth rate and sporulation for each isolate were measured in vitro on PDA plates amended with different concentrations of the fungicides. Plates were arranged in a completely randomized design with three replications per treatment. All fungicides were effective in vitro but there were significant differences in sensitivity among isolates. Bayleton had the highest mycelial inhibition followed by Milraz, while copper oxychloride had the lowest mycelial inhibition rates, ranging from 81% to 88%. However, copper oxychloride was more effective in inhibiting sporulation.The inhibitory effect of each fungicide was concentration-dependent, where twice the recommended concentration had the highest inhibitory effect, followed by the recommended concentration. Our results show that the fungicides used by farmers against C. gloeosporioides, the causal agent for anthracnose, are effective. We, however, recommend further field tests in different avocado-growing areas so as to validate their efficacy against various isolates and under different environments.


Introduction
Anthracnose caused by Colletotrichum gloeosporioides (Penz.)Penz & Sacc.has been reported on a wide variety of crops, such as avocado, almond, coffee, guava, apple, dragon fruit, cassava, mango, sorghum, and strawberry [1][2][3].Colletotrichum gloeosporioides is a significant economic constraint to avocado production worldwide [4].Anthracnose disease can occur on avocado fruits from setting until harvest.The disease results in fruit abortion and may also manifest as black spots on fruits which lower fruit quality [5,6].
The disease inoculum is mainly conidia from diseased dead wood, leaves entangled in the tree canopy, branch terminals, mummified fruits, and flower bracts [7].The conidia are disseminated through rainfall, irrigation, and heavy dew [8].
Disease management includes cultural, chemical, and biological control, along with the use of resistant cultivars [9][10][11][12].Cultural control of anthracnose requires good orchard sanitation procedures.Furthermore, adequate postharvest handling practices must be applied to prevent cuts and bruises to fruit surfaces.Fruits showing symptoms of postharvest disease should not be packed into cartons containing healthy fruits.Biocontrol agents such as Streptomyces sp.A1022 [13], use of food preservatives such as carbonates, bicarbonates, and potassium sorbate [12], heat treatments [14], UV irradiation [15], microwaves [16], chitosan [17], and 2 Advances in Agriculture natural products such as essential oils [18,19] have been used to control the postharvest disease.Chemical control during fruit development is the main strategy to reduce the incidence of postharvest diseased fruits [20].Application of fungicides such as Benomyl, Mancozeb, Metiram, Propineb, Thiabendazole, and Prochloraz has been used for disease management in South Africa, New Zealand, and Australia [21,22].Prochloraz, a nonsystemic fungicide, has been used in the packing line to control postharvest disease development.It affects the mycelial growth of the pathogens and acts as a sterol inhibitor impeding the synthesis of a fatty acid ergosterol, an important component in fungal cell wall development [9,10].
In Kenya, management of anthracnose in avocado has been a challenge due to lack of registered fungicides.Fungicides registered for the management of fungal pathogens in other crops such as Bayleton 25WP (Triadimefon 250 g/Kg), Milraz 76WP (Propineb 70% and Cymoxanil 6%), and Green Cop 500WP (copper oxychloride 500 g/Kg) have been used for anthracnose management in avocado.
Inhibitory effect of Bayleton 25 WP (Triadimefon 250 g/Kg), Milraz 76 WP (Propineb 70% and Cymoxanil 6%), and Green Cop 500WP (copper oxychloride 500 g/Kg) used by farmers to control anthracnose in avocado in Kenya has not been determined.The aim of this study was to provide information on the efficacy of the fungicides against C. gloeosporioides isolates in Kenya with the aim of providing preliminary technical information required for further testing and possible registration of the effective fungicides against avocado anthracnose in Kenya.

Source of Isolates.
Samples of infected fruits were randomly collected from avocado orchards in Murang'a County and taken to the laboratory.The fruits were washed using tap water and blotted to remove excess water.The fruits were surface-sterilized using 0.5% sodium hypochlorite for 30 seconds.Small sections from the edge of necrotic area were cut aseptically and plated on potato dextrose agar (PDA).Hyphal tips of the mycelia were subcultured to obtain pure cultures.Colletotrichum gloeosporioides was morphologically identified based on cultural and microscopic characteristics.Single-spore cultures were preserved on PDA slant universal bottle and stored at 4 ∘ C. A total of 46 isolates were used in this experiment.

Preparation of Fungicides Inhibition Treatments.
In a previous survey, Bayleton 25 WP (Triadimefon 250 g/Kg), Milraz 76 WP (Propineb 70% and Cymoxanil 6%), and Green Cop 500WP (copper oxychloride 500 g/Kg) were identified as the fungicides used by farmers to control anthracnose.The concentrations used were determined based on the manufacturer's recommended doses on the fungicide label.Three concentrations of each fungicide were formulated for use as follows; twice the recommended rate, the recommended rate, and half the recommended rate (Table 1).Fungicide suspensions were prepared by dissolving specific quantities of each fungicide in warm PDA medium after autoclaving.About 15 ml of fungicide amended PDA medium was poured into sterile 9 cm diameter Petri dishes.Each fungicide concentration was replicated three times.Petri dishes with nonamended PDA media served as controls.
Disks of PDA, 5 mm in diameter, with actively growing mycelium of 7-day-old cultures, were cut out with sterile cork borer and placed in the centre of each plate.The amended and control Petri dishes inoculated with mycelia plugs were arranged in a completely randomized design in a sterile hood/chamber at room temperatures of 22-24 ∘ C in the Mycology Laboratory, KALRO, Kandara.The fungi were allowed to grow until mycelia growth covered the entire surface of the control plates.

Evaluation of Fungicides on Mycelia Growth Rate and
Sporulation.After every two days, radial mycelial growth measurements were taken on each plate and recorded.Final measurements were taken after ten days when fungal growth in the control plates fully covered the plates and these measurements were used for data analysis.Inhibition of radial growth due to various fungicidal treatments was computed based on colony diameter on control plate using the following formula as described by [23]: where  is growth in control plate and  is growth in fungicide-treated plate.Further, the plates were flooded with 15 ml distilled water to bring the spores into suspension.Sporulation capacity was determined by counting the number of spores using a hemocytometer.Sporulation was expressed as the total number of spores as determined from 1 ml subsample.

Data Analysis.
Analysis of variance (ANOVA) was used to compare fungicides inhibition percentages (radial mycelial growth and sporulation) using statistical Genstart version 6 software at 95% confidence level.Treatments were separated using Fisher's protected Least Significant Difference (LSD) test.

Results and Discussion
The mycelial growth of C. gloeosporioides isolates was inhibited significantly by the three fungicides' treatments with growth rates reduced by more than 80% (Table 2).Sanders et al. [24] observed similar results, where most of the C. gloeosporioides isolates obtained from diseased avocado fruits were highly sensitive to Prochloraz and Thiabendazole, with no growth being observed.The mycelial growth inhibition from the fungicide treatment significantly increased with an increase in the fungicide concentration (Table 2); however, none of the concentrations evaluated completely inhibited mycelial growth.Similar observations were made by Sanders et al., [24] when C. gloeosporioides isolates were subjected to various concentrations of Benomyl fungicide in vitro.
Among the fungicides, Bayleton had the highest mean inhibition percentages, with copper oxychloride showing the lowest inhibition percentages (Table 2).These fungicides probably acted as a sterol inhibitor impeding the ergosterol (fatty acid) synthesis, which is an important component of the fungal cell wall [12].
Inhibition of mycelial growth could further be explained by the multisite activity of the fungicide such as Milraz (Propineb and Cymoxanil), which inhibits synthesis of nucleic acids, amino acids, and other cellular processes [25].
Although farmers have been using these fungicides on avocado, they are not registered as preharvest chemical treatments for anthracnose on avocados in Kenya.Similarly, farmers have been using Benomyl for the management of anthracnose of avocado in South Africa although it is registered for the control of Cercospora spot of avocado [24].Use of fungicides whose efficacy has not been tested might lead to incorrect application rate, which could favour the development of resistant strains of the causal agent [5].
The mean number of spores recorded from C. gloeosporioides isolates treated with different concentrations of fungicides differed significantly (Table 3).Isolates treated with different concentrations of Bayleton produced a higher number of spores as compared to Milraz and copper oxychloride treatments (Table 4).As the concentration of the fungicide was increased, spore production was significantly reduced but was not eliminated.

Conclusion
All the isolates were sensitive to Bayleton, Milraz, and copper oxychloride in vitro.Bayleton is the most effective fungicide in inhibiting mycelial growth and sporulation of C. gloeosporioides in vitro.

Recommendation
Further field tests should be done in different avocadogrowing areas so as to validate these fungicides' efficacy against various isolates and under different environments.

Table 1 :
Fungicides concentrations used to determine sensitivity of Colletotrichum gloeosporioides isolates from diseased avocado fruits.

Table 2 :
Effect of selected fungicides on mycelial growth of Colletotrichum gloeosporioides isolates.
* Data represent means of three replications.Means followed by same letter are not significantly different at  ≥ 0.05 according to Fisher's protected LSD test.
* Data represent means of three replications.Means within a column and followed by same letter(s) are not significantly different at  ≥ 0.05 according to Fisher's protected LSD test.

Table 4 :
Effect of selected fungicides on C. gloeosporioides sporulation.Data represent means of three replications.Means followed by same letter are not significantly different at  ≥ 0.05 according to Fisher's protected LSD test. *