INVITRO INHIBITION OF RADIAL GROWTH , SPORULATION AND GERMINATION OF ALTERNARIA SOLANI BY SOME FUNGICIDES *

One laboratory study was carried out at the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka to test the efficacy of these three fungicides; Antracol, Benlate and Captan in the control of Alternaria solani with a view of recommending the potent ones to farmers for use in the control of disease caused by Alternaria species. Cultures of A. solani isolated from diseased tomato plants were used for the study. Effects of the fungicides on radial growth and sporulation were investigated. The cultured plates were set up in a completely randomized design in four replicates. Radial growth measurements were determined by taking the diameter of the colony in each cultured plate along two equatorial axes of the plate and their average recorded for all four replicates of a given concentration to the nearest millimeter. Antracol, Benlate and Captan inhibited radial growth, sporulation and germination of A. solani. Captain was the most effective of the fungicides in inhibiting radial growth causing 46.80, 63.29, 68.94 and 73.75 percent inhibition at 10, 100, 200 and 500 ppm respectively. These inhibitions were significantly greater (p= 0.05) than those of Benlate and Antracol at the same levels followed by Benlate while Antracol was the least. Therefore, the percent intibition in vitro clearly showed that among three fungicides tested, Captan was the most effective and has the best potential of being employed in the control of A. solani in the field or in the green house.


INTRODUCTION
Alternaria solani Sorauer, the casual organism of early blight of potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) is a common and frequently serious pathogen of these food crops in most parts of the world where these crops are important (Mathur and Shekhawat, 1986;Agrios, 2005;Chaerani andvoorrips, 2006 andMajeed et al., 2014).Its destructive effect on crops has been recorded from Mexico to Canada in North America, Java, Bermuda, Eastern United States, Pennsylvania and Africa (Keinath et al., 1996;Poya, 1996, Agrios, 2005;Olanya et al., 2009;whiting et al., 2013).It is wide spread in distribution but most important in the warm regions of the world.It is prevalent in cultivated soils and mostly found as saprobe on dead decaying plants and in soil from which the conidia are picked and invade laboratories as contaminants (Agrios, 2005).Infection by A. solani is characterized by the appearance of dark or almost black, more or less circular dead areas or spots upon the leaflets, which show a concentric series of rings giving the lesion a target board effect.Adjacent spots subsequently enlarge and coalesce to cover much surface of the leaf forming more extended dead areas and seriously infected plants suffer premature defoliation (Abdalla et al., 2014;Wiggins, 2014;Chaerani and Voorrips, 2006;Pandey, 2003).According to these authors, the disease causes large economic loss to farmers especially when the season begins with abundant moisture or frequent rains followed by warm and dry weather which are unfavorable for the host and help in rapid disease development.Yield losses up to 79% due to early blight disease have been reported from Canada, India, the United States and Nigeria (Chaerani and Voorrips, 2006).The fungus can cause disease on the foliage (leaf blight), stem (collar rot) and fruit, and can result in severe damage http://dx.doi.org/10.4314/as.v13i1.5 during all stages of plant development.The leaf blight phase, commonly referred to as early blight, is the most important phase of the disease and can result in complete loss of the crop when incidence is severe.A. solani has also been implicated in causing early blight of lettuce (Lactuca sativa L.), eggplant (Solanum aethiopicum) bell pepper and hot pepper (Capsicum spp.) and other members of the Solanaceae (Whiting et al., 2013 andWiggins, 2014).The genus Alternaria contains a number of species that are widely involved in disease of plants, animals and man.For instance A. solani produces a highly phytotoxic antibiotic (alternaric acid) which causes wilting in higher plants and also contaminates human food (Rossman et al., 1990, Hawksworth et al., 1995).They survive on a wide range of substrate and have been isolated from plant sources.Among plants, diseases caused by these species are leaf spot of cabbage and cauliflower by A. brassicola, leaf spot of cucumber and melon by A. cucumerina , black rot of carrot by A. radicina, leaf blight of carrot by A. dauci and rot of citrus fruit by A. citri (Laemmlen 2001;Kucharek 2000 andRossman et al., 1990).In addition, Alternaria species have been implicated in a newly discovered disease of sweet potato (Ipomoea batatas) known as leaf spot and stem blight of sweet potato caused by A. bataticola (Lopes et al., 1994).Control of early blight on crops has been attempted traditionally by use of cultural methods including crop rotation and removal and destruction of infected leaves and stems.While these methods have been helpful to some extent, the level of success achieved has not been adequate to meet the growing demands for these crops.Fungicides treatments are generally the most effective control measures for early blight disease (Chaerani and Voorrips, 2006).Fortunately, some fungicides including Maneb, Zineb, Mancozeb, Chlorothalonil, thiram, Capan, Fludioxonil, Iprodione and Imazalil have been recommended by some authors (Laemmlen, 2001 andWhiting et al., 2013) for control of Alternaria diseases.Reports of effective use of Antacol, Benlate and Captan to control other fungal diseases are well documented (Hamini-kadar et al., 2014;Pscheidt, 2014;Govindappa et al., 2011;Zafar et al., 2010;Raziq et al., 2008;Nawar, 2007;Goertz 2004;Gupa and Aneja 2001;Khokar and Jaffery, 2000;Amadioha, 1998 andCasa et al., 1998).Although the performance of several fungicides against Alternaria diseases has been determined in vitro and in vivo by many researches, there are scanty information on the effects of Antracol, Benlate and Captan on radial growth, sporulation and germination of Alternaria solani.Therefore, studies were carried out in the laboratory at the University of Nigeria, Nsukka, Nigeria to test the efficacy of these fungicides in the control of A. solani with a view to recommending the potent ones to farmers for use in the control of disease caused by Alternaria species.

MATERIALS AND METHODS
Cultures of A. solani previously isolated from diseased tomato plants and maintained on Potato Dextrose Agar (PDA) slants in the Department of Botany, University of Nigeria, Nsukka, Nigeria, were used in this experiment.The fungicides Antracol (Zinc propylene bis-dithio-carbamate), Benlate (Methyl-1-butlyl (carbamoy1) 2-benzimidazole carbamate), and Captan (N-trichloromethyl thio)-4-cyclohexane 1, 2dicarboximide) were also obtained from the same place and used in the experiment.The effects of the fungicides on radial growth and sporulation were investigated on Potato-Dextrose Agar (PDA).The PDA was before use autoclaved for 20 minutes at pressure of 1.1kg/cm 2 ) and temperature (121 0 c).Each fungicide was weighed, suspended in sterile distilled water and added to molten PDA in flask at approximately 50 0 c in proper amounts to achieve final concentration of 10, 100, 200 and 500 ppm (parts per million active ingredients).The media to which no fungicides were added served as the controls.The flasks containing the different fungicides levels and autoclaved media were thoroughly shaken to ensure uniform distribution of the fungicides and 20ml each of the fungicide level was dispensed into each four 90mm Petri dishes.The media after solidifying were inoculated each with a 2mm mycelial disc cut from the periphery of a 7-day old culture of A. solani as described by Ugwuja and Chiejina (2011).The culture plates were set up in completely randomized design in four replicates and incubated for seven days at room temperature (27±4 0 C).Radial growth measurements were done by taking the diameter of the colony in each culture plate along two equatorial axes of the plate and their average recorded for all four replicates of a given concentration to the nearest millimetre.The same plates from which radial growth measurements were taken were used for the sporulation test.After eight days of incubation, each plate was flooded with 8ml of distilled water to form conidia suspension.The conidia were dislodged using sterile camel's hair brush and filtered through a double layer of cheese cloth to remove mycelia fragments.The spore concentration in both the control plates and fungicide amended plates were assessed with the aid of the Neubauer's improved haemacytometer to obtain actual spore amounts and to determine the extent to which each fungicide inhibited sporulation.The effect of the fungicides on spore germination of A. solani was assessed on Potato-Dextrose Broth (PDB).Five millilitre of conidia suspension obtained earlier was centrifuged, the supernatant poured away and the Invitro Inhibition of Radial Growth, Sporulation and Germination of Alternaria Solani residues (spores) used immediately for germination test.Four levels (10, 100, 200, and 500ppm) of each fungicide were made, each level was dissolved in appropriate amount of PDB and 0.05ml each of a fungicide level was placed in each four microscope slide in a plate lined with moistened filter paper.Using the tip of a transfer needle, some 500 conidia were added to each medium on a slide from the spore residues and thoroughly mixed.The mixtures were incubated for two hours at room temperature (27+4 0 C).At the end of this period, germinated spores were counted on each slide by means of a hand operated tally counter.Spores were considered germinated if the germ tube was as long as the spore or exceeds half the diameter of the spore as described by Nene et al. (1979) andMunkvold (1993).On each slide 200 spores were counted and the percentage of those that had germinated determined.All relevant data were subjected to analysis of variance (ANOVA) and mean separation done using Fischer's least significant difference (F-LSD).

RESULTS AND DISCUSSION
Antracol, Benlate and Captan inhibited radial growth, sporulation and germination of Alternaria solani.The effects of the various levels of the fungicides on radial growth of the fungus on PDA after seven days of inoculation are shown in Table (1).The analysis of variance showed significant differences in the treatment means.Percentage inhibition increased as the colony diameter decreased and as the concentrations of the fungicides increased.The colony diameter, a measure of the radial growth was inhibited to various extents by all concentrations of the fungicide tested.Captan was the most effective of the fungicides in inhibiting radial growth causing 46.80, 63.29, 68.94 and 73.75 percent inhibition at 10, 100, 200 and 500ppm respectively.These inhibitions were significantly greater (P=0.05)than those of Benlate and Antacol at the same levels.Benlate was the next in efficacy and caused 20.38, 27.12, 29.43 and 51.77 percent inhibition of radial growth at the same levels while Antracol was the least (Table 1).The effects of the fungicides on sporulation (conidial production) of the fungus on PDA after eight days of incubation are shown in Table (2).Sporulation was similarly inhibited to various extents by all the levels of the fungicides.Captan again was the best of the fungicides in this regard followed by Benlate and Antracol.Although Captan was most effective than Benlate and Antracol, in this regard, the spore concentration under these treatments differed slightly from each other as shown in Table (2).Table (3) shows the effect of the fungicides on spore germination of A. solani on PDB after 2 hours of incubation at room temperature.The percentage germinations and percentage inhibitions revealed that Captan was the most effective of the fungicides causing only two percent germination at 10ppm and no germination at other levels.Benlate was the next in efficacy to Captan followed by Antracol.The results showed that at a lower concentration of 100ppm, Captan gave maximum inhibition of germination.In the overall investigations, the best interaction was achieved by Captan at 500pmm.The use of protectant or eradicant fungicides to control fungal diseases depends on their ability to inhibit germination, growth and sporulation (Mehrotra and Aggarwal, 2003).From the present study Captan exhibited the greatest fungitoxic effect on A. solani.This result corroborates the findings of Ellis et al. (2011); Govindappa et al., (2011); Sugha et al. (1995) working separately on different Fusarium species.However, Zafar et al. (2010) in their studies on mango malformation caused by F. mangiferae found that Captan exhibited minimal effect on colonies of F.mangiferae in vitro test whereas Benlate gave excellent control.The reports of Aveling and Snyman (1993); Biggs et al, (1993) were also at variance with the result of the present study.Filajdic and Sutton (1992) reported that Captan could not control Alternaria blotch of apples caused by A. mali.at variance with that of Raziq et al. (2008), Khokhar and Jaffrey (2000) and Mohammad et al. (1990) who reported that Antracol was highly effective against Peronospora destructor.The observed differences in the performance of these fungicides could be due to detoxification before the site of action has been reached, lack of conversion of a compound into the fungi-toxic principal or the production of inhibitory factor (Cremlyn, 1991).Moreover, various species of fungi show great variation in their ability to resist or tolerate the actions of fungicide in different environments (Agrios, 2005).
Although Benlate achieve limited inhibition of growth, sporulation and germination of A. solani, it has been reported to be a highly effective and broad spectrum systemic fungicide against Fusarium solani, and Rhizoctonia solani (Nawar, 2007).
In our study, radial growth, sporulation and germination of A. solani were inhibited at higher concentration of Antracol.Similar results were observed by Hamini-Kadar et al. (2014), Pathan et al., (2005) and Rajput et al. (2012).However, this result is In view of this, there is need for further studies on these fungicides in the field or green house in order to ascertain their full potential in the control of A. solani.We observed that germination of Alternaria spores was totally inhibited by Captan at a lower concentration of 100ppm.This suggests that Captan can effectively serve as soil drench to control germination of Alternaria spores at that level of active ingredient.The results also indicate that at higher levels (up to 500ppm) Captan can effectively control already developed disease and stop its spread in the field.
In conclusion, therefore, the percent inhibition in vitro clearly showed that among the three fungicides tested Captan was the most effective and has the best potential of being employed in the control of A. solani in the field or in the green house.