ANTIMYCOTIC POTENTIAL ASSESSMENT OF TRICHODERMA SPECIES AND FUNGICIDES FOR SUSTAINABLE MANAGEMENT OF SCLEROTINIA TRIFOLIORUM CAUSING STEM AND CROWN ROT OF TRIFOLIUM ALEXANDRINUM L.

Sclerotinia trifoliorum, the fungal plant pathogen first reported in 2021 from Pakistan on Trifolium alexandrinum L . (Egyptian clover; an annual winter fodder crop), causing Stem and crown rot disease. About 46% to 55% incidence of this disease was recorded on E. clover cultivated in the irrigated tract of the country in 2018-19. This disease is subjecting significant crop losses and drastically reducing growth. An integrated disease management approach employing biological and chemical control was adopted to manage this wide-spreading fungal pathogen. The fungal antagonists, including Trichoderma harzianum , T. longibrachiatum, and T. asperellum Moreover, fungicides, including Thiophanate Methyl, Tebuconazole, Tubeconazole+Emdachloprid, Chlorothalonil+cymoxanil, Azoxystrobin, Pyraclostrobin+Metiram, and Mancozeb+Metalaxyl were tested under in vitro and field conditions. Among Trichoderma species, the best response was achieved by T . harzianum with 80.61% inhibition compared to control. Among concentrations of T . harzianum , the best response was achieved on 1/10 (1.24 cm) with 2.4 average No. of Sclerotia and 66% inhibition. Under filed condition experiments, the data regarding Disease severity in T. harzianum treated trays was 51.7% compared to untreated/control 73.5%. Besides disease control, the application of T. harzianum showed a significant increase in green and dry fodder weight (851 grams with 87 grams of dry weight) than untreated/control (561 grams with 55 grams of dry weight) in the fourth cut. For chemical evaluation, seven fungicides tested at three concentrations under in vitro trials among these Thiophanate methyl (0.5 cm) with 90.7% inhibition were found more effective. Thiophanate Methyl's application significantly reduced the disease severity compared to control plants with disease severity in fungicide-treated trays was 28.7% compared to untreated/control 73.5% and significant increase green and dry fodder weight (931 grams with 92 grams of dry weight) than untreated/control (561 grams with 55 grams of dry weight) in the fourth cut.


INTRODUCTION
Livestock is an integral part of agriculture in Pakistan, contributing 60.6 percent to agriculture and 11.9 percent to the national GDP.The livestock's gross added value was 1,466 billion in 2019-20 (Economic survey of Pakistan, 2019-20).The growing livestock industry requires low-cost and nutritious fodder and feed, but on the contrary, forage cultivation is decreasing (Burki et al., 2005).Trifolium alexandrinum (Egyptian clover) is an annual, multicut winter fodder that belongs to order Fabales and family Febbacae.(Virender and Narwal, 2000;Amanullah et al., 2005).It plays a vital role in the biodegradation of heavy metals such as lead (Pb), zinc (Zn), copper (Cu), and cadmium (Cd) present in soil (Ali et al., 2012).It can be ensiled with cereals at low pH, high lactic acid, and a lower percentage of nitrogen (Mustafa and Seguin, 2003).The feeding of E. clover fodder supplemental concentrates to the lactating dairy animals may produce 10-15 litter milk per day (Naeem et al., 2006).Egypt, India, Pakistan, Australia, Afghanistan, Southern Europe, the USA (California), Turkey, and South Africa are the significant E. clover cultivated countries (Knight, 1985).Pakistan ranks third with 0.71 Million hectares under E. clover cultivation after India (2 Million hectares) and Egypt (1.1 Million hectares) (Muhammad et al., 2014).Various diseases report that attack and reduce the E. clover production.Amongst, Stem and crown rot disease caused by Sclerotinia trifoliorum is a widely spread and severely crop-damaging disease in Pakistan and other countries worldwide (Faraz and Ijaz, 2021;Faraz et al., 2022).S. sclerotiorum is a fungal pathogen affecting about 500 host plants worldwide.Sclerotinia rot is a major hindering to oilseed brassica crop worldwide (Sharma et al., 2016).The Sclerotinia spp.are reported to cause rots in a wide variety of host plants such as Phaseolus vulgaris L. is a bean plant affected by S. sclerotiorum (de Figueiredo et al., 2010;Shaat et al., 2011), Brassica oleracea L. (Cabbage) is a vegetable commonly cultivated worldwide affected by white mold disease caused by S. sclerotiorum (Lib.) de Bary (Elif et al., 2016).S. sclerotiorum is a necrotrophic fungus causing diseases, including stem rot, head rot, white mold, and crown rot on different commercial crops (Kamal et al., 2016).S. sclerotiorum causes disease stem rot of soybean in central regions of USA (Mueller et al., 2002).Once the pathogen is established, their management is challenging; integrating cultural, biological, chemical, and resistant cultivars approaches the best management strategy to control this disease (Sharma et al., 2016).There are many reports on the management of Sclerotinia spp causing various diseases on various crops.As far as biological management is concerned, Trichoderma sp. had the potential to antagonize the Sclerotinia spp under in-vitro and in vivo conditions.(Figueirêdo et al., 2010) However, these fungi' efficacy had limited consistency when applied to control sclerotinia stem rot incidence in canola fields as sustainable management (Kamal, 2016).T. erinaceum, T. koningiopsis, and T. asperellum are also reported as antagonists that entirely inhibit the mycelial growth of S. sclerotiorum (Boat et al., 2018).Elif et al. (2016) proved different strains of B. subtilis (TV-17C, TV-6F, and TV-12H) as biocontrol agents against Sclerotinia.For chemical management, a scenario of fungicides has been tested to control Sclerotinia spp.such as Iprodione, Carbendazim, and Thiophanate Methyl showed promising results (Figueirêdo et al., 2010;Lehner et al., 2015).Fludioxonil had a positive effect on the morphology and physiology of S. sclerotiorum.(Duan et al., 2013).Sumida et al. (2015) analyzed that procymidone inhibits the growth of Sclerotinia.This research study adopted biological and chemical control strategies to devise the appropriate Stem and crown rot disease control strategy.

Sclerotinia trifoliorum
Evaluation of different antagonistic fungi (Table 1) against confirmed fungal pathogen associated with stem and crown rot of E. clover was done using dual culture technique in in vitro conditions.Fungal cultures of antagonists and pathogenic were acquired from FMB-CC-UAF culture collection and revived on PDA culture medium.Seven to ten-day-old 5 mm diameter bits of both antagonistic and pathogenic fungi were placed on a fresh PDA plate opposite axenic conditions.A 5 mm bit of pathogenic fungi were placed on PDA containing Petri plate kept as a control for comparison.Culture plates were incubated in the incubator under 12 hours alternate light and dark periods.The diameter of fungal cultures was recorded daily to evaluate the inhibitory effect.Antagonism effect of Trichoderma spp. was evaluated by using the categories described by Bell et al., 1982 against S. sclerotiorum, and inhibition efficiency were also calculated by using the formula percent inhibition (Faraz et al., 2020).

( )
Where, C = Growth radius in control plate; T = Growth radius inhibited by antagonistic fungi in a dual culture plate.spores/mL concentration) of fungus was inoculated in 300mL potato dextrose broth (PDB) and incubated in a shaking incubator (I 4000, IRMECO, Germany) at 28 ℃ with 180 rpm.Culture filtrate was collected from culture medium after three days of incubation by initially filtered through gauze (8 layers) and a 0.22 µm pore size filter.Culture filtrate was further diluted into three different concentrations 1/10, 1/100, and 1/1000 dilutions of original filtrates in PDA culture medium and poured in Petri plates.PDA culture medium having no culture filtrates kept as a control for comparison.A 5mm disc of pathogenic fungi was placed in the Petri plate center and then incubated at 20±2 ℃ for four days.
Readings of colony diameters in test plates and control plates were recorded and calculated using the formula described by Küçük et al. (2003) and Faraz et al. (2020).
After ten days of incubation number of sclerotia were also recorded.The experiment was performed thrice, and five replicates of each treatment were used.
In vitro evaluation of different fungicides against the Sclerotinia trifoliorum associated with stem and crown rot of Egyptian clover At different concentrations of 100 PPM, 150 PPM, and 200 PPM (Table 2), seven different fungicides were evaluated using poised food technique under in vitro condition to found the most effective fungicide against the fungal pathogen of Stem and Crown rot of E. Clover.Each active ingredient of fungicides was prepared in sterile distilled water and incorporated in PDA medium and poured in sterilized Petri plates under axenic conditions.A 5 mm pathogenic fungal culture was placed in the center of fungicides containing PDA Petri plates, and a bit of culture was placed in the PDA plate without fungicide kept as a control for comparison.Plates were incubated at 18±2 ℃ under alternate light and dark periods.The fungal cultures' diameter was recorded daily to evaluate fungicides' inhibitory effect (Haq et al., 2021).The inhibition efficiency of fungicides was calculated by using the formula described by Vincent (1947).

( )
Where, C = Diameter of mycelial growth of pathogenic fungal culture in the plate without any fungicide; T = Dimeter of mycelial growth of pathogenic fungal culture in fungicides containing PDA Petri plate.3 shows that the colony growth of all the treatments significantly different from each other.Among Trichoderma species, the best response was achieved by T. harzianum (1.13 cm) with 80.61% inhibition, followed by T. longibrachiatum (1.17 cm) and T. asperellum (2.13 cm) with 69.63% and 63.46% inhibition in comparison to control (Figure 1).The colony growth in the control plate was 5.83 cm.

In Vitro Evaluation of Different Fungicides Against S. trifoliorum
The percent inhibition on different concentrations were significantly different from each other.The data presented in the table 5 shows among different fungicides, the best response was achieved by Thiophanate Methyl (0.5 cm) with 90.7% inhibition followed by Tebuconazole (1.0 cm), Tubeconazole + Emdachloprid (1.3 cm), Chlorothalonil + cymoxanil (1.6 cm), Azoxystrobin (1.8 cm), Pyraclostrobin (2.0 cm) and Mancozeb + Metalaxyl (2.1 cm) with 83.3, 78.2, 72.4, 69.0, 66.4 and 63.2 percent inhibition respectively as compared to control (Figure 3).Mean mycelia growth in the control plate was 5.8 cm.The disease severity index was recorded weekly.Green fodder weight and dry weight were also recorded after each cut of E. clover.Table 6 shows that the T. harzianum significantly reduced the disease severity as compared to control plants.Disease severity in T. harzianum treated trays was 51.7% as compared to untreated/control 73.5%.Table 7 shows green and dry fodder weight significantly increased by the application of T. harzianum.Green fodder weight of T. harzianum treated trays was 851 grams with 87 grams of dry weight than untreated/control 561 grams with 55 grams of dry weight in the 4 th cut.

Evaluation of Fungicide (Selected in in Vitro Evaluation) Against Stem and Crown Rot of Egyptian Clover Under Field Condition
Table 8 shows that Thiophanate Methyl's application significantly reduced the disease severity compared to control plants.Disease severity in fungicide-treated trays was 28.7% as compared to untreated/control 73.5%.Table 9 shows green and dry fodder weight significantly increased by the application of Thiophanate Methyl.Green fodder weight of fungicide-treated trays was 931 grams with 92 grams of dry weight compared to untreated/control 561 grams with 55 grams of dry weight in the 4 th cut.Wakil and Ghonim, 2000;Helmy et al., 2001;Mueller et al., 2002;Shaat and El-Argawy, 2011).Use of fungicides and biocontrol agents (especially Trichoderma species) significantly inhibited the disease Sclerotinia stem rot of soybean caused by S. sclerotiorum (Sumida et al., 2015), fluazinam inhibited 100% of the S. sclerotiorum followed by thiophanate-methyl (Costa and da Silva Costa, 2004), fluazinam and procymidone inhibited the S. sclerotiorum in common bean (Vieira et al., 2012;Reis et al., 2010), different fungicides have a different mechanism of action to inhibit the pathogen like procymidone retard the spore germination of S. sclerotiorum (Picinini and Goulart, 2002).Application time of fungicide is a critical factor that reduces the disease incidence and increases the yield (Mueller et al., 2004).Thiophanate methyl, Fluazinam, and procymidone proved effective against the white mold of common beans caused by S. sclerotiorum (Lehner et al., 2015).Fluazinam is a fungicide that could control the diseases caused by Sclerotinia species (Lemay et al., 2002;Matheron and Porchas, 2004;Vieira et al., 2012;Mahoney et al., 2014) harzianum and its culture filtrates at 1/10 dilution reduced the disease severity to 51.7% compared to untreated/control 73.5%.Green and dry fodder weight significantly increased by the application of T. harzianum.Green fodder weight of T. harzianum treated trays was 851 grams with 87 grams of dry weight than untreated/control 561 grams with 55 grams of dry weight in the 4 th cut.Similarly, many plant scientists proved that Trichoderma species had more significance among different fungal antagonists due to their wide range against fungal plant pathogens, including Fusarium, Pythium, Sclerotinia species (Sarma et al., 2014;Steindorff et al., 2014;Woo et al., 2014).T. harzianum had 56.3% inhibition efficiency in dual culture test against S. sclerotiorum cause stem rot in soybeans, and its culture filtrates inhibit the 51.2% (Muthukumar et al., 2011;Zhang et al., 2016).Tancic, 2013 reported that the Trichoderma species has the potential to antagonize the S. sclerotiorum.Seed treatment with Trichoderma species gave a significant increase in seed germination of soybean and plant height (Singh et al., 2008;Joshi et al., 2010;Mukhtar et al., 2012).T. hazianum and T. viridae proved effective biocontrol agents for managing the S. sclerotiorum by antagonizing the mycelium or production of antibiotics.(Chet and Baker, 1981;Papavizas, 1985;Shaat and El-Argawy, 2011).

Conclusion
Sclerotinia trifoliorum causing Stem and crown rot disease (46% to 55% incidence) on T. alexandrinum L. in Pakistan.For management of this disease fungal antagonist and chemicals were evaluated under in vitro and field conditions.Among antagonistic fungi T. harzianum and Thiophanate Methyl's application among chemicals showed promising inhibition of disease.

Figure 1 .
Figure 1.Mean Mycelia growth of S. trifoliorum in the presence of antagonistic fungi Trichoderma species at a different time interval (a) Efficacy of antagonistic fungi Trichoderma species against S. trifoliorum (b).

Figure 2 .
Figure 2. Mean Mycelia growth of S. trifoliorum and average No. of Sclerotia at different concentration of culture filtrates of T. harzianum (a) Efficacy of different concentrations/dilutions of T. harzianum against S. trifoliorum (b).

Figure 3 .
Figure 3. Efficacy of different fungicides at different concentrations against S. trifoliorum.

Table 1 .
Fungal antagonistic Fungi acquired from FMB-CC-UAF culture collection used for evaluation against S.

Table 2 .
Fungicides used against Stem and Crown rot of Egyptian Clover at different concentrations.

of fungal antagonist (selected in in vitro evaluation) against stem and crown rot of Egyptian clover under field conditions
The most effective fungal antagonist evaluated under in-vitro conditions was used to evaluate in the field conditions at their best concentration level.The most susceptible Agaiti cultivar of E. clover evaluated in the varietal response experiment in FMB research area

Evaluation of fungicide (selected under in vitro evaluation) against the stem and crown rot of Egyptian clover under field conditions
Nagarajan et al. (1983))s evaluated under in vitro conditions at their best concentration level were used to evaluate field conditions as seed treatment and spray.A most susceptible cultivar of Egyptian clover evaluated in the varietal response experiment in FMB research area Department of plant pathology, UAF was used for evaluation.Agaiti cultivar's seed was treated with Thiophanate Methyl at Dosage 200 PPM and sown in artificial sick trays soil containing S. trifoliorum FMB, research area Department of plant pathology, UAF.Aerial applications of Thiophanate Methyl were also made.Disease assessment was done using the key ofDixon and Doodson (1974)andNagarajan et al. (1983).Where, N is the total number of plants assessed, and X, Y, Z are numbered in each category

RESULTS In Vitro Evaluation of Antagonistic Fungi (Trichoderma Species) Against S. trifoliorum Causing Stem and Crown Rot
Bell et al. (1982)t al. (1982)categorization T. harzianum and T. longibrachiatum in class 2 (Trichoderma grows and covers 2/3 of the medium surface) and T. asperellum in class 3 (Trichoderma and Sclerotinia colonize each one, half of the medium surface, and none seems to dominate the other).Table

Table 3 .
Mean Mycelia growth of S. trifoliorum in the presence of antagonistic fungi Trichoderma species.

Table 4
. Mean Mycelia growth of S. trifoliorum and average No. of Sclerotia at different concentration of culture filtrates of T. harzianum.Dilutions of T. harzianum Mean Mycelia Growth in cm Average No. of Sclerotia

Table 5 .
Mean Mycelia growth of S. trifoliorum in the presence of fungicides.

Evaluation of Fungal Antagonist (Selected in in Vitro Evaluation) Against Stem and Crown Rot of Egyptian Clover Under Field Conditions Egyptian
clover seeds were treated with the most effective fungal antagonist T. harzianum, and its culture filtrates at 1/10 dilution mixed in soil artificially infested with S. trifoliorum in trays in which E. clover was grown.

Table 6 .
Stem and Crown Disease severity index of Egyptian clover (T.harzianum over control).

Table 7 .
Green and dry fodder weight of Egyptian clover (T.harzianum over control).

Table 8 .
Stem and Crown Disease severity index of Egyptian clover (Thiophanate Methyl over control).

Table 9 .
Green and dry fodder weight of Egyptian clover (Thiophanate Methyl over control).

Consent to participate:
This article does not contain any studies with human participants or animals performed by any of the authors Funding: CAS and Punjab Agriculture Research Board (CAS & PARB) Project No. 951