Efficacy of Different Fungicides and Bio Control Agents Against Fusarium oxysporum, Causal Agent of Potato Dry Rot

Department of Plant Pathology Faculty of Crop Protection, Sindh Agriculture University Tando Jam, Pakistan; shahbazjawed18@gmail.com Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, China; wanghuafeng2012@foxmail.com, chengxialan@qq.com, gooddranam@yahoo.com, hussainarifkaleri@outlook.com, jatoizaheer@gmail.com, kubar.aftabali@gmail.com, mirmohammadnizamani@outlook.com Department of Agronomy Faculty of Crop Protection, Sindh Agriculture University Tando Jam, Pakistan; asifalikaleri2013@gmail.com


Introduction
Potato (Solanum tuberosum L.) belongs to the family solanaceae that includes other 2000 plant species. Tomato It is a good source of iron while their vitamin C contents help in iron absorption. Its consumption is increasing because it is not only cheap but also a rich source of carbohydrates, starch and contributed a lot in the reduction of food shortage globally 1 .
The losses caused by diseases and insects constitute the major constraints that faced by the potato growers worldwide. Apart from the field diseases, postharvest diseases caused considerable economic losses in the quality and quantity of the produced during transport, storage and marketing 2 . Among the diseases the most important wide spread and important caused by pathogenic fungi, affecting tubers and vegetative parts 3 . A number of field and storage disease of potatoes that reduces the quantity and market value are Black scurf 4 , Late blight (Phytophthora infestans) 5 , Early blight (Alternaria solani) 6 , Powdery scab (Spongospora subterrannea) 7 , Wilt disease (Verticillium albo-atrum) 8 , Fusarium dry rot (Fusarium spp.), Silver scurf (Helminthosporium solani), Gangerene (Phoma exigua) 9 , Pink rot (Phytophthora erythroseptica) and Watery wound rot (Pythium ultimum and P. debaryanum) 10 .
One of the main fungal diseases that attack potato is Fusarium dry rot. This problem persists throughout the world. There are many species of Fusarium reported to cause dry rot of potato worldwide 3 . Fusarium dry rot caused by many Fusarium species like F. coeruleum, F. eumartii, F. oxysporum, F. sulphureum and F. sambucinum 11 . It has been estimated that dry rot caused by Fusarium spp. caused 6 to 25% yield losses 12 . Adding to this yield loss, Fusarium spp. are also well known for the toxins production in attacked host and responsible for mycotoxicoses of humans and animals 13,14 . One of the toxins produced by the Fusarium spp. which because dry rots is trichothecene. This toxin is a serious inhibitor of protein synthesis in prokaryote and can cause serious health troubles 15 .
In Turkey, four Fusarium spp. namely F. sambucinum, F. solani, F. culmorum and F. oxysporum are found to be common causes of dry rot of potatoes and have the potential for complete destruction of potatoes in storage 2 . Eighteen percent of tubers arriving at New York markets from 1972-1980 showed symptoms of Fusarium dry rot 16 . While as high as 60% of graded tubers in Scotland was affected by dry rot 17 .
Symptoms of Fusarium dry rot include minute brown areas on the surface of tubers 18,19 , the infected tubers appear wrinkled and rolled tissues from the surface, and rot also creates depressions/ cavity in the surface of the tuber. These affected tissues turn brown, grey or black. When symptoms advances spore masses of blue, white, yellow, purple, black or pink colour may also observe. Seed tubers and potatoes for consumption may not com-pletely. Storage tuber mummifies and ultimately only the dry shell persists 20,21 . Mode of spread is by planting infected tubers or by contaminated soil, as the pathogen is soil borne, airborne or carried in plant residue 22 .
Biological control of dry rot of potato using different antagonists only evaluated for experimental purposes 23 . Antagonistic organisms like Trichoderma spp. and Pseudomonas aeruginosa have been found to be effective management strategy 24 .
Fungicide like Maxim MZ, Tops MZ, and Moncoat MZ may be used for the efficient control of potato dry rot. Many chemicals, including Thiabendazole, may be applied to seed tubers before sowing 25 . Continuous use of same fungicide is the main factor to develop resistance for it as documented in potato dry rot pathogen against Thiabendazole 11 .
Indiscriminate use of chemical pesticides to control various pests and pathogenic microorganisms of crops plants is causing health hazard both in terrestrial and aquatic lives through their residual toxicity 26 . Much attention is being focused on the alternative methods of pest control 27 .
Keeping in view the importance of potato dry rot recent studies carried out to determine the prevalence of potato dry rot at study area and to evaluate the efficacy of different antagonistic agents and fungicides against F. oxysporum causal agent of dry rot of potato.

Isolation and Identification of Causal Fungus
The isolation and identification of fungi was carried out as described by 2 as follows; tubers were washed under running tap water to remove the mud and then air dried. A 6 Shehbaz Jawed, Hua-Feng Wang, Xia-Lan Cheng, Anum Mehmood, Arif Hussain Kaleri, Zaheer Ahmed Jatoi, Asif Ali Kaleri, Aftab Ali Kubar and Mir Muhammad Nizamani mm diameter and 5 mm deep pieces were excised with a cork borer from the affected area of each tuber. The tubers sections were surface sterilized in 5% commercial bleach solution for 1 min. Tuber sections were dried on sterilized paper and plated on Potato Dextrose Agar (PDA). After 5 days of incubation at room temperature under natural light, predominantly isolated fungal colonies developing from the plant material were identified by microscopic observations with the help of literature. Pure culture of fungus was maintained by periodical transfer on PDA plates. Small colony from corner of the fungal growth was picked up with help of inoculation needle and placed on the surface of new freshly prepared PDA plates. Only one disc transferred per PDA plate and incubated at 25°C resulting pure culture were multiplied periodically on new media throughout the study.

Pathogenicity Test of Causal Pathogen
Pathogenicity test of causal pathogen was carried out for the confirmation of disease-causing fungus under in-vitro conditions to prove the Koch's postulates. The procedure was performed according to Peters 28 . The healthy potato tubers of variety "Diamond" were used in this experiment. Tubers appearing uniform in size (100-120 g) were selected for this test. First tubers were washed to remove the surface soil and sterilized by dipping them in 80% solution of ethanol and then air dried. Then the tubers wounded with a cork borer with a diameter of 5 mm to a depth of 5 mm 28,29 . An agar plug (5 mm diameter) containing active growth of F. oxysporum isolates cut from the margin of a 3-day-old cultures grown on PDA and placed into the wound, which was subsequently sealed with the excised plug of tuber tissue. All the wounded potato tubers were wrapped in polyethylene bags and incubated in the dark at 20°C for 3 weeks. As a control, tubers were wounded with the help of cork borer like it's done in treatment then inoculated with only an agar plug without fungus. After three weeks data were recorded on the basis of symptoms development and lesion area were measured in cm with the help of scale.

Evaluation of Different Bio-Control Agents
Different biocontrol agents were obtained From Agriculture Research Institute, Tandojam to evaluate against Fusarium dry rot of potato by dual culture method. Briefly PDA plates were prepared and inoculated by F. oxysporum and selected biocontrol agent aseptically. Both of them were placed at the periphery of Petri plate at equidistance of 2 to 3 cm in opposite direction. Petri plates inoculated with pathogen only served as control. All the plates were incubated at 25°C in incubators. Plates were observed regularly and data on colony growth in cm were recorded with the help of scale and antagonistic nature of the bio agent was recorded. Resulting data on colony diameter was calculated for percent inhibition over control with the help of formula given below: Whereas, C= Growth of pathogen in control plates; T= Growth of pathogen in dual culture plates.
After prolonged storage interactions were assessed using a key based on observations of Dickinson and Boardman 30 as given below: A. Mutually intermingling growth where both fungi grew into one another without any microscopic signs of interaction. Bi. Intermingling growth where the fungus being observed was growing into the opposed fungus either above or below its colony. Bii. Intermingling growth where the fungus under observation has ceased growth and is overgrown by another colony. C. Slight inhibition where the fungus approach each other until almost in contact and a narrow demarcation line, 0.1-2 mm, between the two colonies clearly visible. D. Mutual inhibition at a distance of > 2 mm.

Efficacy of Different Fungicides
The efficacy of different fungicides for controlling F. oxysporum responsible for postharvest infection was carried out with ten different fungicides. For this purpose, Carbendzim, Topsin-M, Mancozeb, Antracol, Gemstar, Scholar, Nativo, Tilt, Score and Radomil were selected and evaluated with four different doses, i.e., 1 ppm, 10 ppm, 100 ppm, 1000 ppm by food poisoning method under in-vitro conditions. The details of fungicides with their company name, active ingredients and brand name are given in Table 1.

Efficacy of Different Fungicides and Bio Control Agents Against Fusarium oxysporum, Causal Agent of Potato Dry Rot
Concentrations given above were prepared by serial dilution method. The required quantity of fungicide was mixed in the medium after sterilization of media. Medium without fungicide were served as control. Before pouring the media were also amended with streptomycin sulphate at 5 ml/L and penicillin at 10 6 units/L to avoid bacterial contamination. Media without fungicide served as control. Equal volume of media i.e., about 15 ml were poured in each Petri dish and inoculated in the centre with a 5 mm disk of F. oxysporum after solidification of media. These plates were incubated as and data were recorded as described above. Growth was recorded daily till any of the plate found full of the growth of F. oxysporum. Finally resulting data was calculated for inhibition percent because of fungicide with the help of formula as follows: Whereas; C = Colony diameter of pathogen in control plates; T = Colony diameter of pathogen in plates amended with fungicide (treatment plate).

Prevalence of Potato Dry Rot
Potatoes showing dry rot symptoms were collected from different markets (Figure 1). Hundred potatoes were randomly examined from each store. The disease found to prevail in 70% of the stored visited with a very low incidence. Average disease incidences of dry rot of potatoes were recorded 3.4%. The isolation and identification of fungi was carried out by tissue isolation methods. Isolations were made with affected potatoes. Total 100 sections were placed over the surface of PDA medium. After incubation of 5 days at 25°C fungal colonies identified by microscopic observations with the help of literature as Fusarium spp. and Aspergillus spp. F. oxysporum was appeared as most predominant fungus with 59% frequency (Table 2). Pure cultures of F. oxysporum were maintained on PDA medium and were multiplied periodically on new media throughout the study. On PDA medium it produced white aerial mycelia became tinged with light purple. From backside of plates dark purple and produces abounded micro and macro conidia, Microconidia were oval to ellipsoid cylindrical while macroconidia long fusoid to falcate in shape with 3 or 4 septa ( Figure 2). These morphological characteristics are similar those described 19,31 .

Effect of Different Bio-Control Agents on the Growth of F. oxysporum
Four different bio-control agents i.e. Paecilomyces lilacinus, Trichoderma harzianum, T. polysporum and P. varioti were obtained from ARI Tandojam and evaluated against

Pathogenicity Test F. oxysporum on Potato Tubers
Pathogenicity test of F. oxysporum was carried out for the confirmation of disease-causing fungus under in-vitro conditions to prove the Koch's postulates. After 3-week incubation, the inoculated potatoes showed wrinkled and rotted symptoms of typical dry rot of potato. The rotted areas of the potatoes were brown, grey, or black and the rot creates depressions in the surface of the tuber. White fungal growth was also apparent on rotted areas. Lesion area after 20 days of inoculation was extended up to 27.10 mm.
Very small lesion were also found to develop in uninoculated control potatoes (1.8 mm), where tubers were wounded with the help of corn borer like it's done in treatment then inoculated with only an agar plug without fungus (Figure 3).

Effect of different fungicides on colony growth of F. oxysporum:
The efficacy of different fungicides against the colony growth of F. oxysporum was checked under laboratory conditions. For this purpose, Radomil, Topsin-M, Antracol, Mancozeb, Carbendzim, Score, Tilt, Scholar, Nativo, and Gemstar, were selected and evaluated with      F. oxysporum by dual culture method. Both of them were placed at the periphery of Petri plate at equidistance in opposite direction. All four antagonistic organisms cause highly significant inhibition in the growth of F. oxysporum which was higher than 60%. Lowest growth of F. oxysporum was found as a result of interaction of P. varioti ( (Figure 7). Antagonistic nature of the bio-agents was recorded with prolonged incubation. Both specie Paecilomyces of shows D type interaction i.e. mutual inhibition of both at a distance of few mm while Trichoderma spp. shows Bii type interaction i.e., F. oxysporum and Trichoderma spp. produces intermingled growth; growth of the F. oxysporum was ceased and overgrown by antagonist (Figure 8).

Discussion
Dry rot potato is one of the main fungal pathogens that attack potato throughout the world. The disease found to prevail in 70% of the stored visited in Hyderabad region with a very low incidence. Average disease incidences of dry rot of potatoes were recorded 3.4%. It has been estimated dry rot caused by Fusarium spp. causes 6% to 25% yield losses 12 .
Symptoms of Fusarium dry rot include minute brown areas on the surface of tubers 18 , the infected tubers appear wrinkled and rolled tissues from the surface, and rot also creates depressions/cavity in the surface of the tuber.
These affected tissues turn brown, grey or black. When symptoms advances spore masses of blue, white, yellow, purple, black or pink colour may also observe.
Seed tubers and potatoes for consumption may rot completely. Storage tuber mummifies and ultimately only the dry shell persists 14,20,21 .
The isolation from diseases tissues revealed the association of Fusarium spp. and Aspergillus spp. F. oxysporum was appeared as most predominant fungus. There are many species of Fusarium reported to cause dry rot of potato worldwide 3 . Fusrium dry rot caused by many Fusarium species like F. coeruleum, F. eumartii, F. oxysporum, F. sulphureum and F. sambucinum Fuckel 11 . Mejdoub-Trabelsi 24 isolated four Fusarium spp. predominantly associated with dry rot of potato. i.e., F. sambucinum, F. oxysporum, F. solani and F. graminearum. On PDA medium it produced morphological characteristic that are similar those described by 19,31 .
Pathogenicity test of F. oxysporum confirmed the virulent nature of the pathogen and produces the similar symptoms of potato dry rot. Author in 32 confirmed the pathogenicity of two F. oxysporum isolates associated with potato dry rot in Colombia. Inoculation with both induced moderate dry rot. Mode of spread is by planting infected tubers or by contaminated, as the pathogen is soil borne, airborne or carried in plant residue 22 . Therefore, emphasis must be given to control dry rot of potato. So far, this disease is managed with the application of fungicides.
Continuous use of same fungicide is the main factor to develop resistance for it as documented in potato dry rot pathogen against Thiabendazole 11 . Therefore, in present study different fungicide determined against F. oxysporum causal agent of dry rot of potato. Radomil, Topsin-M, Antracol, Mancozeb, Carbendzim, Score, Tilt, Scholar, Nativo, and Gemstar, were selected and evaluated with five different doses, i.e., 1, 10, 100, 1000 and 10000 ppm by food poisoning method. All concentrations of fungicides reduced the growth of F. oxysporum as compared to control. However, higher concentrations were more effective than the lower ones. The growth of the test pathogen gradually decreased with increasing concentrations. All the fungicide at 10000 ppm completely stops the growth of test pathogen. Similarly, Piwoni 33 found sixty isolates of F. avenaceum and forty isolates of F. coeruleum sensitive to used fungicide, while eighty-five isolates of sixty eight percent of F. sulphureum and one isolate of F. culmorum were found not sensitive to Thiabendazole. All the Fusarium spp. were sensitive to Imazalil and were pathogenic when inoculated into potato tubers. Yasmin 34 also found Azoxystrobin, Quinoline, Hymexazol and Fludioxonil with inhibitory effect on mycelial growth of F. oxysporum f. sp. tuberosi. IC50 values for each fungicide were also calculated. It greatly varied for each fungicide. Lowest IC50 value was found in case of Topsin-M, Scholar, Antracol and Radomil. Whereas, highest IC50 value was found in Nativo followed 35 tested six fungicides; Carbendazim, Benomyl, Prochloraz, Azoxystrobin, Fludioxonil and Bromuconazole, against F. oxysporum f. sp. lycopersici with seven different concentrations. Prochloraz and Bromuconazole were the most effective fungicides against the pathogen both in vitro and in vivo, followed by Benomyl and Carbendazim. Fungal radial growth was measured and median effective concentration (EC50) values (µg/ml) determined. Biological controls of dry rot with different bio-control agents such as fungi, bacteria, and yeasts have been reported as effective under experimental conditions 23,36 . Four different bio-control agent's i.e., Paecilomyces lilacinus, P. varioti, Trichoderma harzianum and T. polysporum. All four antagonistic organisms cause highly significant inhibition in the growth of F. oxysporum which was higher than 60%. Antagonistic organisms like Trichoderma spp. and Pseudomonas aeruginosa have been found to be effective management strategy 24 . Author in 34 found reduction in colony diameter of F. oxysporum isolated from potato dry rot by T. harzianum, T. viride and T. virens.
Antagonistic nature of the bio-agents was recorded with prolonged incubation. Growth of pathogen was inhibited by Paecilomyces spp. and mutual inhibition of both antagonist and pathogen at a distance of few mm was observed. Whereas in case of Trichoderma spp. pathogen and antagonist produces intermingled growth, growth of the F. oxysporum was ceased and overgrown by antagonist. Similar interaction was also reported 37 . Author in 34 evaluated different Trichoderma spp. against F. oxysporum isolate from potato dry rot i.e., T. harzianum, T. viride and T. virens and studied interaction mechanisms which include disintegration of host cytoplasm and/or alteration into cords and/or coiling around pathogen hyphae.

Summary
Dry rot is considered the most important post-harvest disease that attack potato throughout the world especially for seed production where are store for prolonged duration. The disease found to prevail in 70% of the stored visited in Hyderabad region with very low incidences. Average disease incidences of dry rot of potatoes were recorded 3.4%. Symptoms of Fusarium dry rot include minute brown areas on the surface of tubers. The infected tubers appear wrinkled and rolled tissues from the surface, rot also creates depressions/ cavity in the surface of the tuber. These affected tissues turn brown, grey or black. When symptoms advance fungal spore masses of varying colour may also observed. The isolation from diseases tissues revealed the association of Fusarium spp. and Aspergillus spp. F. oxysporum was appeared as most predominant fungus. Pathogenicity test of F. oxysporum confirmed the virulent nature of the pathogen and produces the similar symptoms of potato dry rot.
So far, this disease is managed with the application of fungicides. Continuous use of same fungicide is the main factor to develop resistance for it as documented in potato dry rot pathogen against Thiabendazole. In present study, different fungicides were tested against causal agent of dry rot of potato. Ten fungicide i.e., Radomil, Topsin-M, Antracol, Mancozeb, Carbendzim, Score, Tilt, Scholar, Nativo, and Gemstar, were evaluated against F. oxysporum with five different doses, i.e., 1, 10, 100, 1000 and 10000 ppm by food poisoning method. All concentrations of fungicides reduced the growth of F. oxysporum as compared to control. However, higher concentrations were more effective than the lower ones. The growth of the test pathogen gradually decreased with increasing concentrations. All the fungicide at 10000 ppm completely stops the growth of test pathogen. IC50 value for each fungicide was also calculated from fungal radial growth at five different concentrations. It greatly varied for each fungicide. Lowest IC50 value was found in case of Topsin-M, Scholar, Antracol and Radomil. Whereas, highest IC50 value was found in Nativo followed.
Four different bio-control agents' i.e., Paecilomyces lilacinus, P. varioti, Trichoderma harzianum and T. polysporum. All four antagonistic organisms cause highly significant inhibition in the growth of F. oxysporum which was higher than sixty percent. Antagonistic nature of the bio-agents was recorded with prolonged incubation. Growth of pathogen was inhibited by Paecilomyces spp. and mutual inhibition of both antagonist and pathogen at a distance of few mm was observed. Whereas in case of Trichoderma spp. pathogen and antagonist produces intermingled growth, growth of the F. oxysporum was ceased and overgrown by antagonist.

Conclusions
Prevalence of potato dry rot in Hyderabad, Sindh, efficacy of different antagonistic agents and fungicides against F. oxysporum causal agent of dry rot of potato are studied. Different Fusarium spp. and Aspergillus spp. were found associated with the collected potatoes with varying frequencies. F. oxysporum was appeared as most predominant isolated fungus with the maximum frequency of 59%. Typical symptoms of dry rot of potato appeared on artificially inoculated tubers with F. oxysporum. Fungal fruiting bodies also appeared on rotted areas after prolonged storage.
In vitro fungal growth test in the presence of different fungicide were performed in order to find out best fungicide. Ten fungicides viz., Radomil, Topsin-M, Antracol, Mancozeb, Carbendzim, Score, Tilt, Scholar, Nativo and Gemstar were evaluated against F. oxysporum with five different doses, i.e., 1, 10, 100, 1000 and 10000 ppm by food poisoning method. Fungal diameter in the Petri dishes was recorded every day till any of the treatment find full of fugal growth. All concentrations of fungicides reduced the growth of F. oxysporum as compared to control. However, higher concentrations were more effective than the lower ones. The growth of the test pathogen gradually decreased with increasing concentrations. All the fungicide at 10000 ppm completely stops the growth of test pathogen.
IC50 value for each fungicide was also calculated from fungal radial growth at five different concentrations, it greatly varied for each fungicide. Lowest IC50 value was found in case of Topsin-M, Scholar, Antracol and Radomil. Whereas, highest IC50 value was found in Nativo followed.
Four different bio-control agents i.e. Paecilomyces lilacinus, P. varioti, Trichoderma harzianum and T. polysporum were tested against dry rot pathogen. All four antagonistic organisms cause highly significant inhibition in the growth of F. oxysporum which was higher than sixty percent. Growth of pathogen was inhibited by Paecilomyces spp. and mutual inhibition of both antagonist and pathogen at few mm was observed. Whereas in case of Trichoderma spp. pathogen and antagonist produces intermingled growth, growth of the F. oxysporum was ceased and overgrown by antagonist.

Recommendation
The results of the present study shows that dry rot of potato prevail in study area but with low incidences. However, to reduce the present incidences and to reduce the further spread management methods should be evaluated. In-vitro amendment of fungicide in culture media inhibits the colony growth of F. oxysporum. Therefore in-vivo application of fungicide for the control of this disease should be evaluated. Reduction in colony diameter of F. oxysporum was observed with the application of used antagonistic fungi. In this connection further studies should be carried out to find out the alternative of fungicide for management of this disease.

Acknowledgment
This study was funded by the National Science Foundation of China (31660055 and 31660074) and a start-up fund from Hainan University [kyqd1633 and kyqd(zr)1840].