Pathogenicity and In vivo Susceptibility Test of Fusarium Isolates on Potato (Solanum tuberosum L.) Using Syzygium aromaticum (Clove)

Introduction

Potato (Solanium tuberosum L.) is the fourth largest food crop after rice, wheat and corn (Farkhondeh et al., 2013) [6] . Potato is a major crop produced in Plateau State and it is known as a staple crop in many countries of the world (Anonymous, 2010). Fusarium is a global disease of importance particularly to potato, which can be transmitted by seed cuts, spores and other modes (Farkhondeh et al., 2013) [6] . For a long time farmers depend on Chemical pesticides for management of pest and disease because of their availability and effectiveness (Sen et al., 2020) [10] . Excessive and inappropriate use of the chemical pesticides has also been cited as a setback to international trade because of the presence of harmful residues in fresh agricultural produce meant for export (Kussaga et al., 2014) [15s] . Worldwide destruction of potato due to disease is high with an estimated economic loss of 3 to 5 billion dollars annually (Eduardo et al., 2007) [4] . Management of diseases are mostly the use of synthetic fungicide though, its usage has detrimental effects (Van de Mortel et al., 2009) [13] . There is an increase in public demand to minimize pesticide residue in potato products resulting in forcing agro-chemical companies and growers to develop safer compounds (Strange, 2003) [14] . Moreover, phytopathogens develop resistance to some synthetic fungicide rendering them ineffective and leading to increased pathogen aggressiveness (Fontem et al., 2005) [7] . The potatoes produced in Jos, Plateau State are predisposed to the development of fungal diseases which may lead to food insecurity, economic deterioration and environmental pollution (Chuwang et al., 2014) [3] . Global losses due to pest and diseases on food crops are large ranging by means from 8.1 to 21% in potato (FAO, 2021) [5] . Alternative crop protection strategy to synthetic chemical is mainly the use of natural products either from plants or microorganisms (Adhikari et al. 2024) [11] . As a result of the difficulties associated with the management of the pathogen using chemicals with respect to environmental challenges, health hazards and persistence of the chemical residue in potato and potato products, it is necessary to employ the use of plant extracts (bio-control) in plant disease management as suitable alternative. Plant extract was selected for this study because of the potentials it possess as an alternative remedy to curb Fursarium wilt of potato. Botanical Fungicide (s) will benefit a lot of small scale farmers since it will reduce the excessive use of synthetic chemicals and the challenges associated with them.

Materials and Methods

Syzygium aromaticum was obtained from Farin Gada market in sterile polythene bag, conveyed to the Department of Plant Science and Biotechnology, University of Jos for identification and voucher number administration: Syzygium aromaticum (UJH000319). The plant was prepared after washing in three changes of sterile distilled running water, air-dried and pulverized. This was followed by extraction using water and ethanol as solvents. The yield was determined using the formula: Yield (%) = Weight of extract ÷ Weight of plant material before extraction X 100 In vitro assay was carried out after isolating Fusarium from soils obtained from three Local Government Areas of Plateau State, known to have cultivated potato for at least three farming seasons; Jos-North, Barkin Ladi and Mangu Local Government using Potato Dextrose Agar (PDA). The Fusarium obtained was preserved until required for use. Agar well diffusion method as described by Amienyo and Onunze (2015) [1] was adopted five concentrations of 500 mg/ml, 250 mg/ml, 125 mg/ml, 62.5 mg/ml and 31.25 mg/ml were dispensed into petri dishes containing Fusarium isolates spread on the surface of PDA. The extracts were applied in clockwise direction and a standard synthetic fungicide was used as positive control. Negative control with Fusarium isolates was grown alongside the treatments. The plates were incubated at 25±2 o C for 5 days in an inverted form after allowing extracts to diffuse into medium, zones of inhibitions were measured using metre rule and results were recorded in mm. In vivo anti-fungal assay was carried out using complete randomized block design in an experimental layout as shown below in triplicates. The experiment was laid out in a 16 treatment combinations of four potato varieties and four concentrations replicated three times 13 41 14 42 16 45 15 44 10 45 9 46 12 47 11 48 4 33 3 34 2 35 1 36 8 37 6 39 6 39 5 40 9 25 10 26 11 29 12 26 1 29 2 30 3 31 4 32 5 17 6 8 7 19 8 20 16 21 15 22 14 23 13 24 16 9 15 N 14 11 13 12 9 13 10 14 11 15 12 16 1 1 2 2 3 3 4 4 8 5 7 6 6 7 5 8

Colors

Red= 500 mg/ml numbers in superscript: Plots Blue: 250 mg/ml Numbers: Pant stand Green= Synthetic fungicide Yellow= Water Pathogenicity test was carried out on the isolates both in vitro an in vivo, Koch's postulate was also conducted to ascertain that causative agent. Extracts of clove with best activity was applied forth-nightly, 6 WAP and infecting the potato plants. 6WAP, the height, number of stems and number of leaves were determined after emergence of the plant and treatment application. Yield of the havested potatoes were determined. Data generated were subjected to 2-way Analysis of variance and conclusions drawn In Vitro Pathogenicity test Result: Yield of extract revealed that aqueous extracts had more yield (6.28%) than ethanolic extracts (2.88%) as presented on Table 1. Antifungal activity was observed at the highest concentration of 500 mg/ml for both aqueous and ethanolic extracts while lowest activities were observed at lowest concentration of 31.25 mg/ml. At 500 mg/ml, the mean diameter of isolates from Jos north, B/Ladi and Mangu had zones of inhibition of 2.13 mm, 8.20 mm and 3.13 mm respectively for aqueous extract while at 31.25 mg/ml, high resistance was observed across all the isolates because no observable inhibition. The activities were more with ethanolic extract of clove as the zones measured were 9.20 mm, 11.20 mm and 12.20 mm for 500 mg/ml. howerver, at 31.25 mg/ml 1.0 mm, 3.30 mm and 2.0 mm for Jos North, Barkin-Ladi and Mangu respectively. The minimum inhibitory concentration and Minimum Fungicidal Concentration (MFC) of the extracts were determined as presented on Table 2. Means followed by the same letter (s) within the same column are not significantly different at 5% level of probability Means followed by the same letter (s) within the same column are not significantly different at 5% level of probability Means followed by the same letter (s) within the same column are not significantly different at 5% level of probability

Discussion

Yield of Plant extract revealed that the extract could be best extracted using water however, ethanolic extract, though less in yield had better activities in the susceptibility testing. This implies that the ethanol used in extraction has the ability to extraction has the ability to extract active compounds that cannot be achievable with water. Fusarium species isolated from soil samples of B/Ladi showed high susceptibility to clove than the standard control drug Emyxyl used. However, the isolates from B/Ladi soil samples did not show positive pathogenicity in vitro. This may imply that the isolates had more effect or pathogenicity with respect to wilting than rotting. Fusarium is known to cause wilting of potato in the field and rot in storage. Against all the incula obtained, ethanolic extract had best/high activities against standard positive control. Though, it revealed that the higher the concentration the better the activity. The antifungal activities with ethanolic extracts created wider zones of inhibition. Microorganisms are regarded as concealed enemies of mankind responsible for causing devastating damages to living organism (Sundar et al., 2013) [12] . The major factor that determines the quality of potato yield is proper and early cultivation. This is even necessary in the era of climate changes, which makes agricultural plant such as potatoes exposed to various stress factors, and can be mitigated using appropriate methods of cultivation. In this study, cultivated potatoes were assessed for emergence at two and four weeks after planting, results revealed there was no significant difference at two and four weeks. Although, when the emergence count was determined, not all the planted potatoes emerged at two weeks after planting however, at four weeks after planting, all the potatoes planted emerged. Leaves were well established at six weeks. Caruso was the only variety that produced flowers (whitish-yellow in appearance), unlike the other three varieties (Connect, Marabel and Jelly) which did not produce any observable flower throughout their growing period. Caruso are said to be resistant to infections unlike Marabel that seems susceptible. This may be as a result of high moisture content. This may be as a result of high moisture content, although cultivar differences in Fusarium resistance are also known to reflect inducible hormone-mediated defence responses (Nguyen et al., 2024) [8] .It is the variety that had the highest vegetative growth while Jelly appeared to be shorter with reduced vegetative growth. Connect and marabel had moderate vegetative growth. Connect having broad and bunchy leaves. The leaves of Jelly were fewer in number, stem short. At six weeks, plants growth had peaked and they were infected with Fusarium spores of which disease was estlablished within 10-14 days after infection (at eight weeks), the plants had shown the symptoms of Fusarium wilt which appeared less than 14 days after inoculation. Barkin-Ladi isolate was most pathogenic on the plants generaly with minimal exception on Jelly and Caruso varietes. Mangu and Jos-North isolates did not show susceptibility to the pathogen on the four varieties used. What was striking was the fact that the Barkin-Ladi isolate in the in vitro pathogenicity test, showed resistance of the potato tubers to the Fusarium isolate however, the reverse was the case under field trial. The potato under Barkin-Ladi was more susceptible to Fusarium wilt compared to the other two isolates (Mangu and Jos-North). The untreated (negative control) was highly susceptible to the pathogen unlike the potatoes that were infected and treated with the extract of Syzygium aromaticum and the standard (positive) control. Potato had started wilting at 8 weeks. Most effect of the pathogen was observed on the leaves while the disease severity progressed toward maturation and harvest. This implies that, the disease must be avoided by all means on farms, early planting is key and practice of crop rotation is necessary. Plants provide a variety of resources ranging from basic to economic importance. Vegetables including potato are known to be affordable and richest sources of calories, minerals, vitamins, dietary fiber hence, referred to as "protected foods". They are necessary to tackle the problems of malnutrition and improve healthy life. Diseases are classified into groups based on the pathogen(s) responsible. In this study, it was observed in a pilot study that Fusarium once it infects the plant, within two weeks, the plant is destroyed depending on the Fusarium species involved hence, they were infected with the pathogen at 6 weeks to allow the plants attain certain level of maturity. The disease incidence rate for B/Ladi was the highest. Severity assessment revealed that Marabel was severely affected. This may be possible due to the high moisture content of the variety compared to the other three, as monitored across the three local government areas under study. The Fusarium isolates varied in their severity assessment, B/Ladi isolate on physical observation exerted more effect than Mangu and Jos-North isolate. This agrees with the report of Nadia (2021) who reported that Fusarium isolated from infected plants tissues varied in pathogenicity this is in conformity with the reports of Ray et al. (2007) [9] . With B/Ladi isolate showing strongest symphtoms of wilting compared to the other two (Mangu and Jos-North). Nadia (2021) also reported a species of Fusarium that is most aggressive (F. oxysporum) among tested Fusarium species such as F. solami and F. sambucirium. Considering the three locations, there are diverse Fusarium species associated with potato in these regions and the list may likely be incomplete.

Conclusion

Variation of the antifungal activity both in vitro and in vivo revealed the possibility of different species of Fusarium isolated from sources they were obtained. Also the in vitro pathogenicity confirm that however, the in vivo antifungal activities of the extracts against the isolate took a little deviation and the Barkin-Ladi isolate which seemed a bit less pathogenic in vitro was highly pathogenic in the field. Syzygium aromaticum has the required potentials of antifungal acivities against Fusarium wilt. This is because, it delayed expression of wilting after infection and as such could be used to develop a biofungicide in managing Fusarium wilt particularly, the ethanolic extract.

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