Bioactivity of an organic farming aid with possible fungistatic properties against some oil palm seedling foliar pathogens

Synthetic fungicides are necessary evil in crop production, their usage cannot be neglected or abandoned but must be alternated/supplemented with other control measures such as cultural, host resistance and biocontrol methods to reduce their detrimental effect on the environment and living organisms. A bioproduct (wood vinegar) was evaluated against oil palm seedling pathogens at CSIR—Oil Palm Research Institute, Kusi at different concentrations and compared with an inorganic fungicide at the manufacturer’s recommended dosage. Disease pathogens were isolated from collected diseased leaf samples and pure cultures were established on cPDA. PDA was amended with wood vinegar ranging from 0 to 3.35% and 0.1%v/v of carbendazim as a positive control. Daily colony growth was measured in two diagonal lengths and averages of day 6 and day 7 were used to calculate the inhibition percentage for both pathogens. 11 mm/day was the lowest average growth rate recorded for 2.68% v/v of wood vinegar and 14.17 mm/day on control plate of Curvularia species. There was no significant difference between 0.1%v/v carbendazim, 2.68 and 3.35% v/v against Curvularia species whilst significantly, there was difference between 0.1%v/v carbendazim and 2.68 and 3.35%v/v of wood vinegar against Pestalotiopsis species.


Results
Inhibitory effect of wood vinegar on mycelial growth. In-vitro evaluation of wood vinegar on mycelium growth of Curvularia and Pestalotiopsis species at different concentrations was promising when colony diameter was measured. 23.35 and 28.56% were the highest inhibition percentage recorded for Curvularia and Pestalotiopsis species, respectively. The highest concentration (3.35% v/v) recorded the highest inhibition percentage of 28.56% which was not statistically different from the concentration (2.68% v/v) for Pestalotiopsis species whilst both concentrations' inhibition percentages were significantly different from carbendazim at 0.1% v/v, which outperformed the best performing concentrates of the wood vinegar. The two highest concentrations of the wood vinegar (2.68 and 3.35% v/v) were not significantly different from carbendazim at 0.1% v/v when tested against Curvularia species (Table 1, Figs. 1, 2). However, a concentration was considered to be inhibitory, if inhibition percentage was ≥ 20%. Figures 5 and 6 show average daily colony growth of the respective pathogens, Curvularia and Pestalotiopsis species. At day one, there was a significant reduction in growth for both pathogens compared to the growth on control plate but there was slight difference in growth in the subsequent days for treated plates, control and Carbendazim amended plates.
Effect of wood vinegar of spore production and spore germination. Wood vinegar had minimal impact on spore production and germination at the highest concentrations (2.68 and 3.35% v/v) (Figs. 3,4) for Pestalotiopsis species but generally caused very low spore production of Curvularia species (Fig. 3). The inhibition activity of wood vinegar on the developmental stages of the pathogens was minimal at the two lowest concentrations as compared to the highest concentrations above (Figs. 3, 4).  Table 2 shows effect of the concentrations on sclerotia production by Curvularia species at different days. Sclerotia produced on carbendazim treated plates were the highest followed by 2.01% v/v to 3.35%v/v of wood vinegar treated plates at day 13. Subsequently, at day 7 there was no observation of sclerotia on any of the treated plates including control plates. Generally, majority of the healthy wounded leaves inoculated with the two pathogens produced similar symptoms observed at the nursery.

Discussion
Wood vinegar, commonly called wood acid, pyroligneous acid is known to contain several organic compounds including acetic acid, acetone, methanol, phenols and ketones which are produced from agricultural, forestry residue samples, bamboo, sawdust, cotton stalk, Chinese fir sawdust, et cetera [14][15][16] . Wood vinegars possess    16 reported that Curvularia species sporulates profusely under harsh condition of high temperatures in or on putative hosts and conidia are easily dispersed in air, an observation made in this research work was production of numerous sclerotia (Table 2). Initially, growth of both pathogens were slow as compared to unamended plate but in the subsequent days, growth rates were somehow similar for treated plates and control plates with slight growth differences (Figs. 5, 6). Arango et al. 17 reported that brown rot fungi including F. palustris secretes oxalic acid and possesses resistance against copper based wood preservatives whilst white rot fungi are more sensitive to natural chemicals extracted from white spruce (Picea glauca), Jack pine (Pinus banksiana), and red pine (Pinus resinosa) cones such as pinosylvin dimethyl ether and the extractives inhibited the growth of white rot fungi (T. versicolor and Phanerochaete chrysosporium), but slightly stimulated the growth of brown rot fungi (Neolentinus lepideus, Gloeophyllum trabeum and Postia placenta) at the 1:1:1 mixture of these compounds 18 . Oramahi and Yoshimura 19 reported that wood vinegar from Vitex pubescens Vahl inhibited white rot fungus, T. versicolor and a brown rot fungus, F. palustris. The phenol content of wood vinegar is likely responsible for its antifungal activity 15,20 . These results are at par with our findings in the present study. The antifungal action of wood vinegar might be due to its effect on fungal enzymatic activity. Kang et al. 21 observed significant growth inhibition by some organic acids such as acetic, malic, oxalic and citric acid, with acetic acid causing the highest inhibition of fungal growth. Kang et al. 21 examined Colletotrichum gloeosporioides, C. coccodes and C. dematium and confirmed an inhibition of respiration by acetic acid as measured in the culture media. Amendment of medium with acetic acid affected glucose utilization but glucose was re-used after the elimination of acetic acid, accompanying with the fungal growth 22 . Kang et al. 21 however, observed increased catalase activity in C. gloeosporioides when hydrogen peroxide was added to the potato dextrose broth medium. But when hydrogen peroxide together with acetic acid was added to the medium, the enzyme activity rather declined slowly with incubation time, inferring that the interruption Table 2. Sclerotia production of Curvularia isolate at selected days on different concentrations of organic farming aid and carbendazim. −Absent. +Present but low. ++Medium. +++High.  According to Lekete et al. 23 , Carbendazim is an effective chemical against Pestalotiopsis species in vitro and is at par with our present finding when compared to wood vinegar (OFA). However, considering its inhibition effect, wood vinegar (OFA) is a promising product with fungistatic properties.

Conclusion
Wood vinegar possesses antifungal properties which inhibited fungi growth at high concentrations due to the acids, phenol and other organic compound in its composition. High levels of wood vinegar concentrations inhibited both Pestalotiopsis and Curvularia species in-vitro.

Collection of diseased samples, isolation and purification of the pathogens. Symptomatic dis-
ease samples were collected from the OPRI nursery, kept in tablet envelopes, labeled and brought to the laboratory. Diseased leaves were washed under running tap water for one minute, they were surface sterilized in 10% (v/v) sodium hypochlorite solution and washed in two changes of sterile distilled water. They were blotted dry and plated on chloramphenicol amended water agar. Emerging fungal growth from peripheries of cultured leaves was sub-cultured onto a half strength cPDA and pure cultures were established for purification purposes.
Pathogenicity test. Ten leaves from healthy oil palm seedlings were collected from the OPRI nursery, washed under running tap water, surface sterilized with 70% (v/v) ethanol, washed in two changes of sterile distilled water. The leaves were wounded with an inoculating needle and inoculated with the Curvularia isolate (KOP-  and Pestalotiopsis isolate (KOP-21-20) separately. Five leaves were inoculated with each pathogen to prove Koch's postulates 23 in a humidified petri dish in the laboratory.

Media preparation and fungicide amendments.
Two percent (w/v) of 250 ml water agar was prepared and amended with 125 mg of chloramphenicol for isolation of disease pathogens. 39 g of Oxoid potato dextrose agar (PDA) was weighed (using Mettler PM 600 electronic balance) into a medium bottle, one litre distilled water was added for the bioassay of the pathogens. 100 ml of PDA was measured separately into seven 250 ml Erlenmeyer flasks and sterilized at 121 °C, 15 psi for 15 min in a Tuttnauer Autoclave. Upon cooling, the medium was adjusted to the required volume and amended with different concentrations of wood vinegar (Control (0), 0.67%, 1.34%, 2.01%, 2.68% and 3.35% v/v) and the manufacturer's recommended rate of carbendazim (0.1% v/v).
In vitro assay of foliar fertilizer against the pathogens. Food poisoning technique 24 was employed to screen the organic product against pathogens. Five mm of actively growing mycelial plug of a seven-day old Curvularia species (KOP-  and Pestalotiopsis species  were centrally placed inversely on each plate medium (five plates per concentration) and incubated at 31 ± 1 °C in a humidified transparent container under diffused sunlight during the day and darkness during the night (Five plates per treatment). Colony diameters were measured daily from the reverse side of plates with a ruler (average of two diagonal measurements per plate). Sclerotia production was scored qualitatively from day 7 to day 14 after incubation. Percentage inhibition was calculated using the formula by Chaurasia et al. 25

Effect of wood vinegar on different developmental stages of Curvularia and Pestalotiopsis species.
After colony diameters were measured for the respective pathogens, six mycelial plugs (5 mm) of Curvularia species per plate (three plates per concentration) were excised along a radius centrally to the periphery of the culture 26 . Acervuli produced by Pestalotiopsis species after 10 days of culturing were harvested from three plates per concentration into 2 ml of sterilized distilled water in Bijoux bottle. The mycelial plugs of Curvularia were teased with a sterilized inoculating needle and shaken for two minutes on a Heidolph REAX 2000 vortex. 20 µl of spores were pipetted onto Weber Scientific International haemocytometer to determine spore count of both pathogens. Spore production was expressed as the number of spores per mm 2 of colony area. Spore germination was determined using a Riddell mount 27 . Three agar blocks per concentration (0, 0 0.67, 1.34, 2.01, 2.68, 3.35 and 0.1 Carbendazim) were used. 60 µl of spore suspension prepared from unamended PDA plates were spread at different locations on each setup. Germination of spore was measured after incubating the setup for 6 h at room temperature in the dark by examining 30 and 100 spores for Curvularia and Pestalotiopsis species, respectively at 100X magnification of an Amscope Compound Microscope (T490B). A spore was scored as germinated if the germ tube had reached at least the full length of the spore (Fig. 7). Experimental design. Completely Randomized Design (CRD) was used for the in-vitro assay. Both transformed (log transformation) and untransformed data were analyzed using GenStat statistical package 12th edition and the means were compared with Fisher's protected Highest Significant Differences at 1% (Supplementary information 1).

Research involving plants.
Permission was granted before the use of oil palm seedlings and they were handled in conformation to institutional and national laws.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.