In vitro evaluation of antibiotic performances on Trichoderma harzianum and some crop infecting fungi

An in vitro experiment was carried out to evaluate the effectiveness of antibiotics at different concentrations on growth and development of Trichoderma harzianum, Phytophthora infestans, Colletotrichum gloeosporioides, Corticium theae and Fusarium oxysporum found in tea plantation. Three samples viz. sample-1 (Validamycin 60% w/w) @ 45, 60 and 75 ppm, sample-2 (Hexaconazole 2.5% w/w+Validamycin 8.5% w/w) @ 55, 82.5 and 110 ppm and sample-3 (Streptomycin 9% w/w+Tetracyclin hydrochloride 1% w/w) @ 50, 75 and 100 ppm were tested. The result showed that Antibiotics have inhibitory effects on T. harzianum. Unsatisfactory performances in terms of per cent growth inhibition (<80) were recorded on crop infecting fungi. C. theae treated with sample-1 @ 75 ppm and C. gloeosporioides with sample-2 @ 110 ppm had shown maximum 25.50 and 54.19 per cent growth inhibition, respectively. The highest 70.53 per cent growth inhibition of C. theae was observed in sample-3 treated @ 100 ppm. Considering the findings it can be recommended not to use above antibiotics with their respective concentrations in plant agriculture for controlling diseases caused by the said fungi.


Introduction
In 1928, Alexander Fleming, a Scottish scientist, identified penicillin, the first chemical compound with antibiotic properties derived from Penicillium fungi (Wikipedia, 2016).In 1942, Selman Waksman and his collaborators in journal articles first used the term antibiotic to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution (Waksman, 1947).Since then the role of antibiotics has expanded from treating serious infections to preventing infections in surgical patients, protecting cancer patients and people with compromised immune systems, promoting growth and preventing disease in livestock and other food animals, including their uses in plant agriculture for controlling diseases (Gelband et al., 2015).
In plant agriculture, several antibiotics have been commercialized for controlling bacterial diseases, especially fire blight of pear and apple caused by Erwinia amylovora and bacterial spot of peach.In USA, 90% of the streptomycin has been using in plant agriculture for control of fire blight (McManus et al., 2002).Streptomycin is currently also registered for fire blight control in Israel, New Zealand, Canada and Mexico.It has been permitted on an emergency use basis, subject to annual review and under tightly restricted conditions in Germany, Austria and Switzerland.Oxytetracycline is used in Mexico and Central America to control E. amylovora on apple, and diseases caused by Pectobacterium spp., Pseudomonas spp.and Xanthomonas spp. on several vegetable crops.Gentamicin is also used in Mexico and Central America to control various bacterial diseases of vegetable crops caused by species of Pectobacterium, Pseudomonas, Ralstonia, and Xanthomonas (Vidaver, 2002).In the 1990s, an attempt to register gentamicin for plant agriculture in the USA was contested, owing to its clinical importance, and the application was withdrawn without prejudice (McManus et al., 2002).Oxolinic acid, a synthetic quinolone antibiotic, is used only in Israel to manage fire blight of pear and related plants, especially in areas where E. amylovora is resistant to streptomycin (Shtienberg et al., 2001).Oxolinic acid also is registered in Japan for management of bacterial panicle blight of rice, caused by Burkholderia glumae (Maeda et al., 2004;Nandakumar et al., 2009).In rice production, seeds and plants during panicle emergence or flowering are treated with oxolinic acid (Maeda et al., 2004).
Some multinational pesticide companies come up with antibiotics like validamycin, streptomycin and tetracycline hydrochloride for controlling fungal diseases of crops.Validamycin, a non-systemic antibiotic with fungistatic action, produced from fermentation of streptomyces hygroscopicus var.limoneus used effectively for controlling the diseases caused by Rhizoctonia solani in rice, potatoes, vegetables, strawberries, tobacco, ginger and other crops; damping-off diseases of cotton, rice and sugar beet, etc. (Thomson, 1982;NIOSH, 1993;Anon., 1994;Meister, 1994;Tomlin, 2000).Streptomycin is a human antibiotic drug which also is used to control bacteria, fungi, and algae in crops (EPA, 1988).It is obtained by fermentation of Streptomyces griseus, isolated as sesqui sulfate.It has systemic action and effectively used against the diseases especially those caused by gram-positive species of bacteria (Tomlin, 2000).Tetracyclines are a group of broad-spectrum antibiotics whose general usefulness has been reduced with the onset of antibiotic resistance.Tetracycline hydrochloride is used only to treat or prevent infections that are proven or strongly suspected to be caused by susceptible bacteria (Klajn, 2001).
Global consumption and demand for antibiotics to control diseases continue to rise.More dependency on antibiotics accelerates to develop antibiotic-resistant mechanism in the pathogen(s) that is recognized as a major threat to control bacterial diseases and infections worldwide (Stockwell, 2012;CDC, 2013;Gelband, 2015).When a pathogen became resistant to an antibiotic, a new antibiotic with potent active ingredient is introduced in the market that causes more access to antibiotics and expenditure.
However, indiscriminate and overuse of chemical pesticides for controlling pests without prescription, and their adverse effects on food chain, human health and environment are common phenomena.So, introduction of antibiotics in crop husbandry for controlling fungal diseases, their effectiveness against pathogen(s) and residual effects in treated crops have claimed special attention from the researchers.Beside these, no works have been done on antibiotics for controlling fungal diseases in plant agriculture in Bangladesh.In view of the points, the present research work has been undertaken to evaluate the effectiveness of antibiotics on T. harzianum, a beneficial fungus and some crop infecting fungi viz.P. infestans (late blight of potato, damping-off of seedlings), C. gloeosporioides (die-back, anthracnose, rot of fruits, vegetables and other crops), C. theae (black rot of tea) and F. oxysporum (wilt, damping-off, die-back, foot and collar rot of vegetables, etc.).

Materials and methods
The experiment was carried out in the laboratory of plant pathology, Bangladesh Tea Research Institute (BTRI) during October 2014-September 2015.Three samples containing antibiotic were tested on T. harzianum, a beneficial and some crop infecting fungi viz.P. infestans, C. gloeosporioides, C. theae and F. oxysporum found in tea plantation.Potato dextrose agar media (PDA), necessary glassware and equipment required for isolation and culturing fungi were sterilized in advance by autoclave at 121°C temperature for 22 minutes at 15 PSI.

Isolation of fungi
1. Dilution plate method T. harzianum, a beneficial fungus having antagonistic behavior against some harmful fungi and P. infestans, a late blight of potato pathogen that was found surprisingly in tea ecosystem were isolated from the subsoil (0-9" depth) following dilution plate method.In this method, 1g of sun-dried soil known as working sample was taken in a test tube containing 9ml of sterile water.The test tube was shaken thoroughly to obtain a uniform stock solution.Now, 1 ml of stock solution was transferred to a test tube containing 9 ml of sterile water and shaken thoroughly to make 1:10 dilution.Similarly, a serial dilution of 1:1,000 was prepared by transferring 1 ml of soil suspension to two consecutive test tubes.Finally, 1 ml of soil suspension from 1:1,000 dilutions was spread over the agar medium and then shaken smoothly for few seconds.The agar plates were kept in controlled condition for observing the fungal growth.

Tissue planting method
Tea infecting three fungi species viz.C. gloeosporioides (die back of tea); C. theae (black rot of tea) and F. oxysporum (gall of tea) were isolated from infected plant materials using tissue planting method.In this method, infected sample was clean with sterile water and cut into 1.5-2 mm pieces containing both healthy and diseased part.The inocula were then surface-sterilized by using 30% ethanol for 2 minutes and washed three times with consecutive changes of sterile water.Excess water remained onto the inocula was removed by sterilized blotting papers.Four inocula were plated in each of five petri dishes containing PDA media.The whole operation was done in a laminar air flow cabinet.The petri plates were incubated at 29±1 0 C and kept under observation for 2 weeks.

Preparation of antibiotic concentrations
Three samples containing antibiotic viz.sample-1 60 SP (Validamycin 60% w/w) @ 45, 60 and 75 ppm, sample-2 11 WP (Hexaconazole 2.5% w/w+Validamycin 8.5% w/w) @ 55, 82.5 and 110 ppm; and sample-3 10 SP (Streptomycin 9% w/w +Tetracyclin hydrochloride 1% w/w) @ 50, 75 and 100 ppm were tested on isolated fungi.Concentrations in ppm were calculated on the basis of their doses i.e. 75, 100 and 125 g/1,000 liter of water/ha for sample-1 and 500, 750 and 1,000 g/1,000 liter of water/ha for sample-2 and sample-3, respectively.The lowest doses of all antibiotics were regarded as expected/supplied commercial doses.In the laboratory, calculated volume of antibiotic was added with 20 ml of melted PDA medium maintaining the respective concentrations of each.The poisoned growth medium was then poured in the sterilized Petri dish.

Inoculation of fungi into poisoned growth media
Previously isolated and cultured fungi were inoculated into the poisoned growth media separately.Three replications were maintained for each concentration along with their control plates.Inoculated plates were then incubated for further observations.

Observation and data collection
Observations of fungal growth on poisoned media were made every 24 hours of incubation.Hyphal growth extension (diameter) was measured by millimeter scale.Growth inhibition was calculated when the whole control plate (90 mm) covered by fungus and then expressed in per cent by using the following formula: Where, 'A' is defined as growth in control plate and 'B' means growth in poisoned medium.Finally data were analyzed by statistical program using MSTAT C (version 2.10) with the help of computer.
concentrations on test fungi.Earlier study had shown that all fast-growing species (except Pythium ultimum) were efficiently inhibited but not completely suppressed by validamycin (Laar, 1982).
In 2000, Tomlin reported that validamycin applied as a foliar spray, soil drench, seed dressing, or by soil incorporation, at 1.25 g/ha (liquid) for controlling Rhizoctonia solani in rice, potatoes, vegetables, strawberries, tobacco, ginger, and other crops; damping off diseases of cotton, rice, sugar beet, etc. Hexaconazole, a fungicide mixed with an antibiotic validamycin introduced in the market for controlling fungal diseases of crops.But this formulated product had shown unsatisfactory performance on fungi at  Streptomycin and Tetracyclin hydrochloride are basically antibacterial compounds used as human drug commercially.Streptomycin also is used as a pesticide, to combat the growth of bacteria, fungi, and algae (EPA, 1988;Wikipedia, 2016).This compound is extensively used in controlling fire blight of apples and pears (50-100 ppm), crown gall of rose (50-200 ppm), bacterial spots of peppers and tomatoes (200 ppm), wildfire and blue mold of tobacco (100-200 ppm), soft rot and blackleg of potatoes (100 ppm) in North America (Anon., 2006).Combined formulation of streptomycin with tetracycline hydrochloride-a broad-spectrum human antibiotic, for controlling fungi was found to ineffective at different concentrations in the present investigation.The impractical performances of such antibiotics might be due to their inappropriate active ingredients mixed with the formulated products or unwise guidelines prescribed by the marketing companies to convince the growers though their antibiotics are not effective in controlling fungal diseases of crops.

Conclusion
The in vitro results showed that antibiotics have inhibitory effects on T. harzianum.Unsatisfactory performances in terms of per cent growth inhibition (<80) were observed on crop infecting fungi.So, considering the findings it can be recommended not to use tested antibiotics with their respective concentrations for controlling plant diseases caused by the said fungi.Further researches on effectiveness of antibiotics, impacts on human health and environments, residual effects on test crops, and growing antibiotic-resistant in pathogenic fungi should be carried out before introducing them in plant agriculture.

Table 1 .
Evaluation of antibiotic performances on Trichoderma harzianum and some crop infecting fungi