Antifungal activity of crude extracts of Ageratum conyzoides and Chromolaena odorata for management of Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae through in vitro evaluation

Lasiodiplodia is an important genus of fungi causing destructive diseases on perennial crops, including cocoa. Two crucial species of Lasiodiplodia that cause diseases in cocoa are Lasiodiplodia theobromae and Lasiodiplodia pseutheobromae. A variety of weeds is the potential to be applied as botanical fungicides to control the pathogens. The main objective of this study was to evaluate Ageratum conyzoides and Chromolaena odorata leaf extract to inhibit the growth of L. theobromae and L. pseudotheobromae on a synthetic medium. Solvent organic was methanol for weed extraction with a ratio of 1:5. The experiment was conducted through the poison food technique method, both in the solid and liquid medium in three different concentrations, 1, 3, and 5%. The result showed that A. conyzoides and C. odorata were significantly inhibited the colony growth of both Lasiodiplodia in all concentrations in a solid medium. A. conyzoides performed better than C. odorata in all concentrations of both Lasiodiplodia in inhibition. A. conyzoides 5% performed well to suppress the colony growth of L. pseudotheobromae (100%), followed by A. conyzoides 3% and A. conyzoides 1%. A. conyzoides 5% able to inhibit the colony growth of L. theobromae until 100%, followed by A. conyzoides 3% and 1%. Meanwhile, A. conyzoides and C. odorata extract tested on PDB medium at 1, 3, and 5% reduced the fungal biomass significantly at all concentrations. C. odorata was found most effective in inhibiting fungal biomass of both pathogens either on wet weight or on dry weight at 1, 3, and 5% %. A. conyzoides and C. odorata can manage the growth of L. theobromae and L. pseudotheobromae through in vitro conditions.


Introduction
Cocoa (Theobroma cacao L), a source of chocolate, is frequently attacked by a range of plant fungal pathogens wherever it is planted [1]. Together with major quantities of milk, sugar, almonds and peanuts, it supports a multi-billion dollar chocolate industry [2,3]. Among the wide range of important fungal pathogens that affect cocoa production are members of the Botryosphaeriaceae [4-9]. Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae are a member of botryosphaeriaceae, causes diseases such as leaf necrosis, dieback, and canker on many tropical plants [4][5][6][7][8][9]. In cocoa, one of the diseases is caused by Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae is cocoa dieback. The disease is considered a threat to cocoa production in Sulawesi, Indonesia [8,9].
As a newly emerging disease, options of the control method remain limited against the pathogen. Providing a range of effective and efficient control methods is urgent to inhibit the pathogens. Recently, microbial antagonists and fungicides have been tested in suppressing the pathogen growth and incidence [10,11]. Meanwhile, Some of the plants are able to produce secondary metabolites that can act as antifungal compounds [11][12][13][14].
Utilization of local resources for controlling the pathogen is considered more effective and efficient, one of them is weeds around the cocoa farm such as Ageratum conyzoides and Chromolaena odorata. A. conyzoides or better known as goat weed has antifungal properties which can inhibit the growth of microorganisms [15,16]. C. odorata, commonly called kirinyuh or siam weed, is a member of the Asteraceae family that is considered a competitor of the cultivation plants. Antifungal actvity of C. odorata extract against a range of plant pathogenic microbes has been performed by several previous studies [17][18][19][20].
This study aimed to evaluate the antifungal activity of A. conyzoides and C. odorata against Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae through in vitro conditions. The research will provide knowledge about the possibility of using weed extracts for controlling Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae.

Materials and methods
The experiments were conducted in the laboratory at the Department of Plant Pests and Diseases, Hasanuddin University and Agricultural Quarantine Major Service of Makassar.The pathogen isolates of L. pseudotheobromae and L. theobromae were collected in the laboratory at Department of Plant Pests and Diseases, Hasanuddin University 2.1. Preparation and extraction of A. conyzoides and C. odorata A. conyzoides and C. odorata leaves were collected directly from the field. After that, the weeds were washing with tape water thoroughly, then leaves were sundried for three days, crushed and stored in a glass container. The crushed leaf materials of the two test plant species were soaked in methanol (1:5 w v-1) for seven days. After that, extracts were filtered through cloth. The organic solvent was evaporated using a vacuum rotary evaporator. During the process, A constant temperature and pressure were set at 50 O C and 90 rpm, respectively. The extract was transferred in amber bottles and stored in the refrigerator.

In vitro inhibition on mycelial growth in PDA medium
A mycelial-agar plug of 9 mm-diametre of the pathogen was taken from two-day-old culture and placed off the center of the petri dish contains potato dextrose agar (PDA) medium added with plant extracts on each concentration. There are three concentrations, namely: 1, 3, and 5%. The desired concentration of each plant extracts was mixed thoroughly in 20 ml of PDA. Controls were only PDA medium without extracts. Petri plates were wrapped and incubated at 25-27 °C. Each treatment was replicated four times. Observations were taken at 1-day intervals until the mycelia of L. pseudotheobromae and L. theobromae were covered thoroughly on control.
Colony diameter was measured after 24-hour and 48-hour of inoculation by using measurement, 3 and the percentage of inhibition of mycelial growth was calculated as the following formula [21]. (1) Where C = diameter of the colony in check (average of both diagonals, T = diameter of the colony in treatment (average of both diagonals).

In vitro inhibition on fungal biomass in PDB medium
A mycelial disc of L. pseudotheobromae and L. theobromae were taken from the tips of two days old fungal culture use a 9-mm cork borer and transferred to a 40-ml bottle. There is three concentration of each plant extract viz., 1, 3, and 5% were added to potato dextrose broth (PDB) medium. Controls were treated with no extracts. The bottles were wrapped and incubated at room temperature and prepared on the shaker for seven days [22]. Each treatment was replicated four times. After 7-day the fungal biomass in each bottle was filtered out through filter paper and weighed for wet weight on an electric scale. Then, the fungal biomass was dried at 60 °C in the electric oven for two days and weighed for dry weight on an electric scale. Per cent inhibition of fungal biomass was calculated as the following formula [21]. (2) where C = Biomass of the colony in check, T = Biomass of the colony in treatment

Statistical Analysis
The data were analyze by factorial analysis of variance (ANOVA). The mean comparison was made using Tukey's test at 0.05 level of significance.

Results
There is an effect of the plant extracts on mycelia growth of L. pseudotheobromae and L. theobromae both on PDA and PDB medium where the influence was a range from significant to highly significant on 24-hour after inoculation and 48-hour after inoculation whereas concentration effect was also significant to highly significant at 24-hour after inoculation and 48-hour after inoculation. However, the interaction (plant extracts × concentration) effect was mostly not significant in any evaluation event.
Among three concentrations, the concentration of 5% was found significantly superior over other concentrations. The concentration 1% of C. odorata was the least effective in inhibiting the mycelial growth of L. pseudotheobromae and L. theobromae. The concentration of 5% of C. odorata was found significantly inhibite over two other concentrations. The concentration 1% of A. conyzoides was the least effective in inhibiting fungal biomass of two species of Lasiodiplodia, both wet weight and dry weight.  . The dieback disease on cocoa is considered a future threat in cocoa in Sulawesi. As an emerging disease, control method needs to be explored, including using local plant extracts which are easy to access by cocoa farmers. To evaluate the possibilities of plant extracts as an effective control were tested in vitro against L. pseudotheobromae and L. theobromae.
All plant extracts at 1, 3, and 5% concentrations were found statistically effective to inhibit mycelial growth and fungal biomass over control. Both A. conyzoides and C. odorata can suppress fungal growth in any concentration with different inhibition. Either A. conyzoides or C. odorata have been studied for their antifungal properties against different fungi [15][16][17][18][19].
This research is the first effort to evaluate A. conyzoides and C. odorata against L. pseudotheobromae and L. theobromae isolated from cocoa. The plant extracts can inhibit the mycelia growth and fungal biomass of the pathogens. However, extraction of chemical compounds of the extracts and testing on the plant would be better for supporting the in vitro result.

Conclusion
Inhibition of L. pseudotheobromae and L. theobromae through in vitro is influenced by plant extracts and concentrations. A. conyzoides and C. odorata are proved effective in reducing mycelial growth and fungal biomass.