Suppression of Stem-End Rot on Avocado Fruit Using Trichoderma spp. in the Central Highlands of Kenya

Demand for organic avocado fruits, together with stringent food safety standards in the global market, has made producers to use alternative, safe, and consumer-friendly strategies of controlling the postharvest fungal disease of avocado fruits. This study assessed the in vitro efficacy of Trichoderma spp. (T. atroviride, T. virens, T. asperellum, and T. harzianum) against isolated avocado stem-end rot (SER) fungal pathogens (Lasiodiplodia theobromae, Neofusicoccum parvum, Nectria pseudotrichia, and Fusarium solani) using a dual culture technique. The Trichoderma spp. were also evaluated singly on postharvest “Hass” avocado fruits. Spore suspension at 5 × 104 conidial/ml of the Trichoderma spp. was applied on the avocado fruits at three time points, twenty-four hours before the fungal pathogen (preinoculation), at the same time as the fungal pathogen (concurrent inoculation), and 24 hours after the fungal pathogen (postinoculation). In the in vitro study, T. atroviride showed the highest mycelial growth inhibition against N. parvum (48%), N. pseudotrichia (55%), and F. solani (32.95%), while T. harzianum had the highest mycelial growth inhibition against L. theobromae. Trichoderma asperellum was the least effective in inhibiting the mycelial growth of all the pathogens. Similarly, T. virens showed the highest mycelial growth inhibition against N. pseudotrichia at 45% inhibition. On postharvest “Hass” fruits, T. atroviride showed the highest efficacy against N. parvum, N. pseudotrichia, and F. solani in all the applications. Trichoderma virens and T. harzianum were most effective against all the pathogens during postinoculation, while Lasiodiplodia theobromae was best controlled by T. virens, T. harzianum, and T. asperellum during postinoculation. Both T. atroviride and T. harzianum present a potential alternative to synthetic fungicides against postharvest diseases of avocado fruits, and further tests under field conditions to be done to validate their efficacy. The possibility of using Trichoderma spp. in the management of SER on avocado fruits at a commercial level should also be explored.


Introduction
Avocado (Persea americana Mill.) is one of the economically most important subtropical fruit crops worldwide and a major foreign exchange earner in Kenya [1,2]. In the year 2017, 300 MT of avocado fruits were exported from Kenya, contributing USD 50.5 million to the GDP [3]. Globally, avocado fruits are cultivated in a wide range of agroecological zones for both domestic and commercial purposes [4]. e fruit is valued worldwide for its high nutrition value due to the presence of monounsaturated fatty acids, several minerals (potassium, iron, and phosphorus), and vitamins (E, B, and C), as well as lipids and phytochemicals.
Moreover, the consumption of avocado fruit is associated with improved overall diet quality [2,5].
Stem-end rot (SER) disease causes losses of avocado fruits in all avocado-growing regions of the world. e disease affects the fruits during marketing, storage, or even transit to the market [6]. Members of the Botryosphaeriaceae family (Diplodia mutila, D. pseudoseriata, D. seriata; Dothiorella iberica; Lasiodiplodia theobromae; and Neofusicoccum australe, N. nonquaesitum, and N. parvum) have mainly been associated with SER on avocado fruits. Other pathogens reported to cause the disease include Colletotrichum gloeosporioides or C. fructicola and Diaporthe foeniculacea Phomopsis perseae, yronectria pseudotrichia, Dothiorella aromatica, Pestalotiopsis versicolor, Rhizopus stolonifer, Fusarium sambucinum, and Fusarium solani [6][7][8][9]. In a previous study [10], L. theobromae, N. parvum, N. pseudothrichia, and F. solani pathogens were identified as the leading cause of SER of avocado fruits in Kenya. Over the years, synthetic chemicals have successfully been used to control plant diseases, and they have a promising future. However, chemical residues on produce, nonbiodegradable toxins on fruits and soil, and the high cost of the chemicals have continued to be of significant concern [11]. Additionally, consumers are increasingly demanding reduced use of chemicals on produce. More so, food safety standards and organic food consumer organizations demand minimum detectable residues in produce [12]. Utilizing microbial fungicides, microbial antagonists, and biocontrol agents (yeast, bacteria, and antagonistic fungi) offers a potential alternative to synthetic fungicides in the management of postharvest diseases of fruits [13]. A biological control approach involves using microorganisms to reduce or maintain the postharvest fungal pathogens below economic loss [14].
Currently, several postharvest diseases of fruits can be controlled by either natural microbial antagonists or artificially introduced microbial antagonists [15]. Microbial antagonists present several advantages over synthetic fungicides. ey are environmentally friendly, safer in application, have nontoxic residues, and are economical to produce [16]. Trichoderma spp. have been widely used during postharvest storage to protect fruits and vegetables of commercial importance such as chilli, mangoes, apples, bananas, strawberries, and tomatoes [17,18]. Trichoderma viride, T. harzianum, and T. koningii have demonstrated antagonistic activity against L. theobromae and Colletotrichum musae that cause postharvest crown rot disease complex of banana stored at room temperature and at cold storage [19]. Trichoderma harzianum has also been reported to control anthracnose in bananas, maintain postharvest fruit quality, and reduce natural fruit infections [20]. Substantial progress has been made towards biological control of postharvest diseases of avocado fruits [16,21]. However, no attempt has been made towards the biological control of postharvest disease of avocado fruits in Kenya.
is study, therefore, investigated the antagonistic activity of the selected Trichoderma spp. against fungal pathogens associated with stem-end rots of avocado fruits in the central highlands of Kenya.

Materials and Methods
2.1. Source of the Isolates. Samples of "Hass" avocado fruits were obtained from orchards and local markets in Murang'a County in the central highlands of Kenya. e fruits were incubated at room temperature (22°C-25°C) at Kenya Agricultural and Livestock Research Organization (KALRO), Kandara, for 7-14 days to allow development of stem-end rot disease. Fruits that displayed stem-end rot symptoms were cleaned with clean tap water, surface-sterilized by dipping in 75% ethanol for 3 minutes, and rinsed in distilled water. Small pieces of rotten tissues from the margins of the rot were aseptically isolated, inoculated on potato dextrose agar (PDA), and incubated at room temperature (22-25°C) for 5 days. Pure cultures were obtained by subculturing the hyphal tips of the mycelia. e isolates were identified based on morphological and cultural characteristics and confirmed through molecular identification. Slant universal bottles were used to store the pure cultures in PDA at 4°C. Four commonly isolated pathogens were used in this study.

Source of the Antagonists.
Two commercial species spp. (T. asperellum and T. harzianum) and two locally acquired spp. (T. atroviride and T. virens) of Trichoderma were used in this study. Trichoderma harzianum was obtained from the biological fungicide TRIANUM P (T. harzianum Rifai strain T22, 1 × 10 9 colony-forming units (cfu)/gram of dry weight) from Koppert Biological Systems. Trichoderma asperellum was obtained from the biological fungicide MAZAO SUS-TAIN (TRC900 1.7 × 10 9 cfu/gram of dry weight) from real IPM. Trichoderma atroviride (KRI) and T. virens (BMLT54P1) were obtained from the Department of Agriculture Science and Technology, Kenyatta University. Spore suspension was prepared by flooding fourteen-dayold pure cultures in PDA with sterile distilled water. A sterile wire loop was used to scrape off the conidia and bring them to suspension. e suspension was then filtered through a double-layer muslin cloth, and the collected filtrate was diluted serially to 1 × 10 5 . A haemocytometer was used to adjust the spore concentration.

Antagonistic Activity of Trichoderma spp. against Avocado
Fruit Stem-End Rot Pathogens In Vitro 2.3.1. Dual Culture Assay. e inhibitory activity of four Trichoderma spp., T. atroviride, T. virens, T. asperellum, and T. harzianum, against the four SER fungal pathogens, Lasiodiplodia theobromae, Neofusicoccum parvum, Nectria pseudotrichia, and Fusarium solani, was determined using the dual culture technique [8]. Sterile PDA was poured into Petri dishes 9 cm in diameter. e mycelial disc (5 mm in diameter) from the edge of actively growing 7-day-old fungal colonies was placed at the edge of one side of the Petri dish. A mycelial disc 5 mm in diameter from an actively growing Trichoderma spp. culture was placed at the opposite edge of the Petri dish. e Petri dishes inoculated at one edge with a mycelial disc 5 mm in diameter of fungal pathogens served as control. Each treatment was replicated 6 times, and the Petri dishes were incubated at 25 ± 2°C. e mycelial growth of the test pathogen and of the antagonist was recorded. Percentage inhibition was calculated using the following formula as described by Rajendiran et al. [22]: 2 Advances in Agriculture where C-mycelial growth of the pathogen in control and Tmycelial growth of the pathogen in the dual-culture plate.

Effect of Trichoderma spp. against Stem-End Rot Fungal
Pathogens on Postharvest Avocado Fruits. Mature "Hass" avocado fruits were harvested from a farm in Murang'a County. Fruits with no apparent signs or symptoms of a disease and no physical damage were selected. e fruits were washed with running tap water and surface-sterilized by dipping them in 75% ethanol for 3 minutes. e fruits were then rinsed with distilled water and placed on sterilized trays to air-dry at room temperature. e ability of Trichoderma species to suppress the development of SER on "Hass" avocado fruit was tested by adding each of the antagonists at three time points: (i) 24 hours before the fungal pathogen (preinoculation), (ii) at the same time as the fungal pathogen (concurrent inoculation), and (iii) 24 hours after the fungal pathogen (postinoculation) [8].
"Hass" avocado fruits were individually sprayed at the stem end with 50 µL spore suspension (5 × 10 5 conidial/ml) of the SER fungal pathogens (L. theobromae, N. parvum, N. pseudotrichia, and F. solani). A similar quantity of the antagonist was also used, and each of the treatments was replicated four times. e pathogens and the antagonist were applied on the fruits according to the schedule mentioned above. Fruits inoculated with each pathogen only and replicated four times served as the control. e experiment was conducted twice. e inoculated avocado fruits were placed in sealed plastic containers (separate container for each fruit) at 25 ± 2°C and incubated. Evaluation was conducted after 12 days by cutting the fruits lengthwise. A category scale of 0 to 5 was used to rate the severity of SER development on the avocado fruits; Table 1. e percent disease index was calculated using the following formula as described by Lakshmi et al. [23]:

Data Analysis.
e data obtained were recorded and tabulated in a spreadsheet. After that, the data were exported to Min Tab 17.0 software (Minitab, LLC). Descriptive statistics were generated upon which the data were expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) was used to analyze the statistical significance of difference among treatment groups. Tukey's post hoc test was used for pairwise separation and comparison of means. e hypothesis for significance was tested at p ≤ 0.05.

Effect of Trichoderma spp. on the Severity of SER on
Postharvest "Hass" Avocado Fruits. All Trichoderma spp. inhibited the development of SER on avocado fruits. Fruits treated with T. asperellum in the three inoculations (preinoculation, concurrent inoculation, and postinoculation) remained free from SER caused by F. solani. Similarly, the severity of SER by L. theobromae was significantly different (p ≤ 0.05) reduced up to 10%, 7.5%, and 5% in the three tests, respectively. During the three inoculation, T. asperellum reduced SER on avocado fruits by N. parvum up to 30%, 55%, and 40%, respectively. ere was no development of SER by N. pseudotrichia during concurrent inoculation with T. asperellum; however, during preinoculation and postinoculation, SER severity reduced to 20% and 7.5, respectively (Table 3).
All fruits remained free from SER due to N. parvum, N. pseudotrichia, and F. solani during concurrent and postinoculation with T. atroviride. Trichoderma atroviride did not inhibit development of SER on the fruits by L. theobromae during concurrent and postinoculation; however, during preinoculation, the fruits remained free from SER development due to N. pseudotrichia. Similarly, the severity of SER due to L. theobromae, N. parvum, and F. solani was reduced to 5%, 7.5%, and 7.5%, respectively, during preinoculation with T. atroviride (Table 3). During postinoculation with T. harzianum, no SER developed on the avocado fruits. Besides, during concurrent inoculation of T. harzianum with N. parvum, N. pseudotrichia, and F. solani, the fruits remained free from SER. Trichoderma harzianum did not inhibit development of SER on the avocado fruits due to L. theobromae during concurrent inoculation and N. pseudotrichia during preinoculation (Table 3).
All fruits remained free from SER when Trichoderma virens was inoculated 24 hours after the fungal pathogen. Similarly, during preinoculation, the avocado fruits remained free from SER due to N. pseudotrichia and F. solani, while in concurrent inoculation, no SER developed on the fruits due to N. parvum and F. solani. e severity of SER due to L. theobromae was reduced up to 42.5% in both preinoculation and concurrent inoculation with T. virens (Table 3).
Trichoderma atroviride was most effective in controlling the development of SER by N. parvum, N. pseudotrichia, and F. solani in all treatments, while Trichoderma virens and T. harzianum were most effective during postinoculation (Table 3).

Discussion
e ability of T. harzianum to significantly inhibit the mycelial growth of L. theobromae reported in this study agreed with the study by Wijeratnam et al. [24] where T. harzianum was reported to effectively control L. theobromae that caused SER of papaya and mangoes in Sri Lanka. Similarly, Bhadra et al. [25] reported the greatest inhibition of T. harzianum against L. theobromae in concurrent inoculation. Moreover, T. harzianum has been reported to significantly reduce stem-end rot of Rambutan caused by L. theobromae [26].