MANAGING BOTRYTIS BLIGHT I N GREENHOUSE ORNAMENTALS THROUGH HOST RESISTANCE AND BIORATIONAL PRODUCTS By Sunil Shrestha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pa thology Master of Science 20 20 ABSTRACT MANAGING BOTRYTIS BLIGHT IN GREENHOUSE ORNAMENTALS THROUGH HOST RESISTANCE AND BIORATIONAL PRODUCTS By Sunil Shrestha Botrytis cinerea , an airborne necrotrophic fungus is one of the most important and destructive p athogens of greenhouse - grown ornamental crops. The pathogen incites leaf, stem, and flower blight reducing plant quality and marketability. The overall goal was to provide grow ers with disease management options by combining host resistance and biorational products. Geranium and petunia cultivars were screened for resistance and biorational products were evaluated for efficacy against B. cinerea B . cinerea. In contrast , only few products prov ided effective control in . In , diseas e assessment indicated that B acillus amyloliquefaciens , Pseudomonas chlororaphis , Aureobasidium pullulans , and e xtract of Swinglea glutinosa provided a level of efficacy similar to the fungicide standard fenhexamid. S . glutinosa res ulted in protection similar to the fenhexamid across both trials. petunia had significantly higher disease severity and AUDPC values than ly less disease than unia showed that A . pullulans and G liocladium catenulatum effectively limited disease similar to fenhexamid. Host re sistance could reduce fungicide inputs and be used in combination with biorational controls for effective Botrytis blight control. iii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Mary Hausb eck for giving me opportunity for pursuing higher studies u nder her supervision and for her mentorship and guidance . I also would like to thank my committee members Dr. Jan Byrne and Dr. Timothy Miles for their support and advice for the completion of my m I am grateful to Blair Harlan for his help and assistance in the research activities and also like to thank fellow graduate students and all the members of the Hausbeck lab for their support and encouragement during my studies here. Finally, I would like to thank my parents and loved ones for alw ays being there and for continuous support and encouragement throughout my life. iv T ABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ v LIST OF FIGURES ................................ ................................ ................................ ..................... vi LITERATURE REVIEW ................................ ................................ ................................ ............ 1 INTRODUCTION ................................ ................................ ................................ ......................... 2 SIGN AND SYMPTOM S ................................ ................................ ................................ ............. 4 EPIDEMIOLOGY ................................ ................................ ................................ ........................ 4 GROWTH IN CULTURE MEDIA ................................ ................................ ............................. 6 DISEASE ASSESSMENT ................................ ................................ ................................ ............ 7 DISEASE MANAGEMENT ................................ ................................ ................................ ........ 8 CULTURAL CONTROL ................................ ................................ ................................ ............. 8 BIOLOGICAL CONTROL ................................ ................................ ................................ ......... 9 CHEMICAL FUNGICIDES ................................ ................................ ................................ ...... 11 FUNGICIDE RESISTANCE ................................ ................................ ................................ ..... 12 CULTIVAR RESISTANCE ................................ ................................ ................................ ....... 13 LITERATURE CITED ................................ ................................ ................................ .............. 16 CHAPTER 1. EVALUATION OF GERANIUM CULTIVARS AND BIORATIONAL PRODUCTS TO CONTROL BOTRYTIS BLIGHT IN GREENHOUSE ........................... 23 ABSTRACT ................................ ................................ ................................ ............................. 24 INTRODUCTION ................................ ................................ ................................ ................... 25 MATERIALS AND ME THODS ................................ ................................ ........................... 27 RESULTS ................................ ................................ ................................ ................................ . 34 DISCUSSION ................................ ................................ ................................ .......................... 48 LITERATURE CITED ................................ ................................ ................................ .............. 52 CHAPTER 2. MANAGEMENT OF BOTRYTIS CINEREA IN PETUNIA USING CULTIVAR RESISTANCE AND BIORATIONAL PRODUCTS ................................ ........ 58 ABSTRACT ................................ ................................ ................................ ............................. 59 INTRODUCTION ................................ ................................ ................................ ................... 60 MATERIAL AND METHODS ................................ ................................ .............................. 62 RESULTS ................................ ................................ ................................ ................................ . 67 DISCUSSION ................................ ................................ ................................ .......................... 74 LITERATURE CITED ................................ ................................ ................................ .............. 78 v LIST OF TABLES Table 1. Geranium species and cultivars evaluated for susceptibility to Botrytis cinerea ..............3 1 Table 2. Biorational products and a standard fungicide evaluated for efficacy against Botrytis cinerea on gerani um .............................................................................................................. .. .......3 2 Table 3. Mean number of blig hted leaves, foliar lesions and leaves with sporulating B. cinerea on geranium cultivars 20 days follow ing inoculation .........................................................................3 5 Table 4. Area under disease progress curve (AUDPC) values for blighted leaves, foliar lesions and leaves with sporulating B. cinerea on geranium cultivars when ino culated ...................................3 6 Table 5. Mean number of blighted leaves, foliar lesions and leaves with sporulating B. cinerea on following inocu lation ....................................................................................................................3 9 Table 6. Area und er disease progress curve (AUDPC) data for blighted leaves, foliar lesions and leaves with sporulating B. cinere products ............................................................................ ............................................................. 40 Table 7. Mean number of blighted leaves, foliar lesions and leaves with sporulating B. cinerea on days following inoculation .................................................................................... ........................4 4 Table 8. Area under disease progress curve (AUDPC) data for blighted leaves, foliar lesions and leaves with sporulat products ............................ .............................................................................................................4 5 Table 9. Biorational products a nd a standard fungicide evaluated for efficacy against Botrytis cinerea on petunia ............................. ..................................................................................... . .......6 5 Table 10. Disease severity on petunia cultivars in the greenhouse observed 21 days following inoculation with Botrytis cinerea ................................. ..................................................................6 8 Table 11. Area under disease progress curve (AUDPC) for disease severity on petunia cultivars in the greenhouse when inoculated with Botrytis cinerea ..................................... .............................6 9 Table 12. petunia when inoculated with Botrytis cinerea and treated with biorational products and a fungicide standard ..................... .................................................................................................... ................ 7 2 vi LIST OF FIGUR ES Figure 1. Highly susceptible (A,B) and moderately resistant (C,D) g eranium cultivars observed 20 days after inoculation with Botrytis cinerea A: Ringo 2000 Violet, B: Maverick Scarlet Picottee, C: Pinto Premiun . . .............................................................3 7 noculated with Botrytis cinerea and treated with biorational products A: Untreated inoculat ed control, B: Streptomyces lydicus (Actinovate), C: Pseudomonas chlororaphis (Zio), D: Extract of Swinglea glutinosa (Ecoswing), E: Aureobasidium pullulans (Botecto r), F: Fenhexamid (Decree). ... ........................................... ...4 2 Figure 3. cinerea and treated with biorational products. A: Untreated control, B: Pseudomonas ch lororaphis (Zio), C: Bacillus subtilis (Serenade Opti), D: Aureobasidium pullulans (Botec tor), E: Gliocladium catenulatum (Prestop), F: Fenhexamid (Decree). ..................................................................4 7 Figure 4. Highly susceptible (A, B) and least susceptible (C, D) petunia cultivars observed 21 days following the ino ......................................... ........... ..................... 70 when inoculated with Botrytis cinerea and treated with biorational products A: Untreated control, B: Bacillus subtilis (Serenade Opti), C: Streptomyces lydicus (Actino vate), D: Ulocladium oudemansii ( BotryStop), E: Pseudomonas chlororaphis (Zio), F: Aure obasidium pullulans ( Botector), G: Gliocladium catenulatum ( Prestop) , H: Fenhexamid (Decree) . .. ......................................................... ............... .................... 7 3 1 LITERATURE REVIEW 2 INTRODUCTION Ornamental production is an important agricultural enterprise in the U.S.; plants may be grown in the field/outdoor shaded areas or in greenhouses (Daughtrey and Benson, 2005) . The value of floricu ltural crops in the U.S. for the 6,386 growers who produce a reven ue of $10,000 or more, was nearly $4.63 billion in 2018. The total production area included 859 and 39.3 million m 2 for covered and greenhouse space, respectively ( USDA, National Agricultural Statistics, 2019) . Michigan ranks third in the U.S., behind California and Florida, in the production of floriculture crops, accounting for 10% of the total wholesale value of $467 million in 2018. In the same ye ar, there were 569 floriculture crop producers in the state with $10,000+ in sales with a total of 4.45 million m 2 of greenhouse space ( USDA, National Agricultural Statistics, 2019) . Michigan leads the nation in production of flats of seeded geraniums, petunias, begonias and impatiens; hanging baskets of geraniums from either seed and v egetative cuttings, petunias, begonias, impatiens and pan sies/violas; and potted geranium, petunias, peony and Easter lilies ( USDA, National Agricult ural Statistics, 2019) . The wholesales value for product ion of flats of g eraniums (vegetative cuttings) in the U.S. in 2018 was $7.6 million with total sales of $1.15 million in Michigan just behind California with sales of $3.84 million. The total wholes ale value of hanging baskets and geranium pots ( vegetativ e cuttings ) w ere $30.56 million and $81.64 million respectively in 2018, Michigan being the highe st producer in nation with sales value of $7.47 million for hanging baskets and $12.45 million for ger anium pots. Similarly, p etunias ha ve a total wholesale va lue of $141.7 million when sold in 2018 as flat, pots and as hanging baskets in the U.S; the sales value in Michigan was $31.3 million. Michigan ranks first for the production of geranium (17%) and p etunia (22%) with the highest total wholesale values thro ughout the nation ( USDA, National Agricultural Statistics, 2019) . 3 Disease management is a c oncern of ornamental crop growers as marketing depends on the aesthetics of the plant (Daughtrey and Benson, 2005) . Greenhouse - grown ornamentals are susceptible to Botrytis blight or grey mold disease (Hausbeck and Moorman, 1996) incited by Botrytis cinerea (telemorph: Botryot i nia fuckeliana ), an airborne necrotropic fungi belonging to S clerotiniaceae family on Helotiales order under Ascomycota division (Whetzel, 1945) . Considered to be one of the most destructive pathogens both pre - and post - harvest (Dean et al. , 2012) , i t caus es damping - off, stem canker, blossom and leaf blight, and bud, stem , crown and blossom end fruit rot (Williamson et. al., 2007 , Jiang et. al., 2018 ) . Other diseases include damping - off of young seedlings, leaf spot and root rot of corms, rhizomes, tubers, seeds (Hausbeck and Moorman, 1996) . Globally, m ore than 200 crop species are affected by B. cinerea includin g ornamental plants, vegetables and fruits (Moyano et al., 2004; Williamson et al., 2007 ; Hahn, 2014) . The average cost to protect crops from this pat hogen (cultural measures, fung icides, biocontrol) is a pproximately $51.98 /h a with a global expense of approximately $ 1 .3 billi on annu ally (Steiger, 2007 ; Dean et al., 2012). Average protection costs against B. cinerea var y between $19.5 /ha for pumpkin in China to more than $169 /ha for citr us in Japan ( Steiger, 2007) . The c ost of limiting B. cinerea in grape represents 50% the total market value. However, the pathogen also cause s noble rot in grape b unches used to produce valuable sweet wines (Dean et al., 2012) .The cost of controlling this pathogen is 5% of total botrytis market for ornamentals , bulb vegetables and leafy vegetables, 7% in cucurbits and 9% in solanaceous crops (Steiger, 2007) . B . cinerea is problematic all over the world ranging from tropical and subtropical to temperate cold regions and can remain active at the tem perature of 0 0 c which makes it an important pathogen even during storage and shipping (Elad et al., 2007 ) . It infects crops 4 growing in both the greenhouse and field, causing crop damage whe n conditions favor disease (Elmhirst et al., 2011) . Production of vegetables and ornamentals in the greenhouse s favor s grey mold as warm temperatures, high relative humidity, free moisture, and a lack of air exchange provide favorable environmental conditions for the pathogen (Elad and Shtienberg, 1995; Paulitz and Belanger , 2001) . SIGN AND SYMPTOMS Grey mold sympto ms and signs include w ater - soak ed tissue, necrotic spots, soft rot , and powdery grey conidial masses on the surface of infected tissue (Will iams on et al., 2007) . In some cases, tiny , round , black resting spores called sclerotia may form on infected tissue. B. cinerea reproduces on dead , decaying host tissue and organic matter and sporulates producing grey conidia (Punja and Utkhede, 2003) . I nfe ction may be initiated on dead flowers and then spread to other tissue . Infection may occur via conidia that germinate and infect susceptible tissue or from mycelium growing from infected to healthy tissue (Moorman and Lease, 1992) . EPIDEMIOLOG Y B . cinerea is an ubiquitous fung us that infects the lea ves, flowers, and fruits of ornamentals, small fruit crops and vegetables (Elad and Shtienberg, 1995) . It may survive in the short term as mycelium, conidia or chlamydospores o r for longer periods as sclerotia (Holz et al., 2007; Wil liamson et al., 2007) . The pathogen produces large amount of conidia in the asexual cycle which serve as primary inoculum. Sclerotia are the primary structures for pathogen survival which germinate primarily by produc ing conidiophores. Germination of scl erotia is favored by low temperature with the optimum temperature of 5 0 C. Sclerotia may germinate and produce apothecia in the field to initiate the sexual cycle but the apothecial stage is rarely found 5 for most of the Botrytis species including B. cinerea (Coley - Smith, 1980 ; Hahn, 2014) . Conidia are the primary inoculum with optimum germination occurring at 20 0 C. The optimum tempera ture for infection is between 15 to 25 0 C. (Jarvis, 1989) . Temperature influences the germination of conidia and lesion development and can occur between 4 and 25 0 C; germination is inhibited at 30 0 C (Salina s et al. , 1989) . The optimum temperature for conidial germination is 22 to 25 0 C with RH > 90% . Relative humidity of 100% for 5 hrs is suff icient for disease infection at room temperature (Salinas et al., 1989) . Th e wet and humid conditions in the greenhouse from misting during propagation promotes conidial germination and expansion and coalescence of lesions which reduces plant quality (Hausbeck and Pennypacker, 1991b) . Conidia are oval or globose one - celled hyaline structures produced by conidiophores and borne in clusters on short sterigmata (Pande et al., 2002) . They are short lived and influenced by temperature, light, moisture and microbial activity (Holz et al., 2007) . In some Botrytis species, the septate and brown mycelium can survive for rela tively l onger periods in bulbs and other vegetative parts and can overwinter as mycelium in the bark and buds of infected grape vines. (Coley - Smith, 1980) . Botrytis cinerea is a problem in the storage and shipping of geranium cuttings as conidia are deposited on the plant ce and may infect and cause disease during the environmental conditions associated with shipping (Hausbeck and Pennypacker, 1991b) . Conidia are dispers ed from infected plants when there is a rapid decline in rela tive hum idity which often occurs mid - morning. When there is rapid fluctuation in the relative humidity, the conidia are released through a hygroscopic mechanism (Jarvis, 1989) . Maximum conidial dispersal occurs when the relative humidity fluctuates rapidly bet ween 85 and 65%; vigorous hygroscopic movement of the conidiophore occurs with a 5% change within this range (Ja rvis, 1960) . The peak atmospherical conidial concentration among geraniums in a greenhouse 6 was associated with grower activity including watering, fertilization, pesticide application and harvesting cuttings (Hausbeck and Pennypacker, 1991 a , 1991 b ) . Conidial dispersal within the greenhouse is influenc ed by the magnitude of previous dispersals; a high concentration occurring on one day may be followed by a reduced concentration the following day (Hausbeck and Moorman, 1996) . B. cinerea conidia can be carried by the insect Drosophila melanogaster on its c (Louis et al., 1996) The geranium foliage infected with B. cinerea was greater than that of petunia and impatiens when inoculated and incubated under similar environmental conditions (Pritchard, 1 995). Sp orulation incidence when geranium leaves were inoculated was high for one - wk - old leaves, declined when the leaves were 4 wks old and increased when leaves were 4 - to 10 - wks old (Sirjusingh et al., 1996) . B. cin erea sporulated more rapidly in one and 10 - wk old leaves at 25 0 C when leaves were wet for 8 24 hr (Sirjusingh and Sutton, 1996) . Sporulation of the pathogen on inoculated geranium flowers increased when the wetness duration increased from 8 to 24 h at 15 0 C and from 4 to 6 h at 30 0 C . Sporulation was more efficient and prominent when conidia were inoculated directly to leaves compared to petals of geranium (Sirjusingh and Sutton, 1996) . GROWTH IN CULTURE MEDIA The maximum germination of B. cinerea conidia occurred at 20 0 C after 24 hrs of incubation on potato d extrose agar (PDA) media. B. cinerea mycelial growth on PDA media at 98 - 100% RH for 24 hr increased up to 20 0 C, but decreased rapidly above 25 0 and died at 35 0 C 7 ( Ahmed et al., 2014 , Van Den Berg and Lentz, 1 968 ) . Botrytis convulata grow s on PDA when incubated for 5 to 7 days at 24 0 C in darkness and then exposed to conti nuous white fluorescent light for 6 days or until colonies are covered with conidiophores (Maas, 1969) . Prune extract lactose yeast extract agar (PLY) is used to culture B. allii ; g rowth increases from 5 0 C t o 20 0 C but is slowed above 30 0 C with n o growth at 35 0 C (Alderman and Lacy, 1981) . The opti mum temperature for mycel ial growth of B. cinerea on potato sucrose agar (PSA) medium was 24 - 28 0 C with s porulation observed at 24 0 C after 3 days of inoculation and reach ed a maximum 4 and 6 days after inoculation (Shiraishi et al., 19 70 b ) . Conidia germinat e in the range o f temperature 5 - 32 0 C but at 10 0 C germination is delayed with only 60% conidi a germinat ion within 48 hrs with an optimum temperature of 20 - 30 0 C. (Shiraishi et al., 19 70 a ) . A selective medium for growth and sporulation of B. cinerea known as Botrytis selectiv e medium (BSM) has been prepared by Kritzman and Netzer ( 1978 ) that contain s fungicides and tannic acid resulting in brown pigmentation after oxidization indicating the growth of Botrytis . DISEASE ASSESS MENT Disease may be assessed based on the number of necrotic leaves and sporulation of B . cinerea . S coring is based on the total diseased l eaf area on each plant with visual scale rating of 0 to 10 where 0 = no lesions, 1= lesions with 1 - 10% leaf area c overed, 2= 11 - 20%, 3= 21 - 30%, 4= 31 - 40%, 5= 41 - 50%, 6= 51 - 60%, 7=61 - 70%, 8= 71 - 80%, 9= 81 - 90% , and 10= 91 - 100% of the leaf area affected b y t he pathogen (Elmhirst et al., 2011) . Köhl et al. (1998) assessed plants in the greenhouse based on the area (%) covered with conidiophores of B . cinerea ranging from >0 to 1 at an interval of 0.1. The disease severity indicates the number of leaves covered with B . cinerea sporulation (spore producing leaf area) and is estimated using the formula: Severity= 8 , where i= number of plants (1 to n), m i = no. of sporulated leaves, P ij = proportion of jth leaf on plant i which have sporulat ion (Köhl et al., 1998) . DISE ASE MANAGEMENT Effective management of B . cinerea in the greenhouse requires an integrated approach including manipulation of the environment, biocontrol agents, and fungicides (Jarvis, 1 989) . Hausbeck and Pennypacker (1991a) suggested that applying fu ngicides or modifying the greenhouse environment should be timed immediately after the harvest of cuttings from geranium stock plants. Sanitation, use of a photo - selective greenhouse covering, heating sys tems, and timely application of fungicides are impo rtant tools to manage B . cinerea in greenhouses (Hausbeck and Moorman, 1996) . CULTURAL CONTROL Botrytis cinerea common ly occurs in the greenhouse where the relative humidity may be high. Reducing the relativ e humidity in the greenhouse can be achieved by ve nting and heating (Hausbeck and Moorman, 1996) . Removing diseased, dead plant tissue, providing proper air circulation , increasing plant spacing, and avoiding plant wounds is necessary (Hausbeck et al., 1996) . Bot rytis cinerea can be managed using several manageme nt strategies but keeping the atmosphere dry is important (Gerlagh et al., 2001) . Reducing moisture in the greenhouse can be achi eved by installing a heating system under the bench, using plastic mulch on top of the pots in a stock plant scenario, and reducing plant density (Hausbeck and Moorman, 1996) . Combining plastic mulch and an under the bench heating system reduced pathogen spo rulation on stock plant leaves more effectively than the single tr eatments. Forced heated air was more effective in 9 reducing disease incidence than the plastic mulch (Hausbeck et al., 1996) . BIOL OGICAL CONTROL Biological control is used predominantly as a preventive disease control measure and is generally not used post infection (Jacometti et al. , 2010) . Generally, biocontrol refers to the use of microbial organisms and natural product extracts that supp resses plant pat hogens and limits disease (Pal and Gardener, 2006) . Biocontrol agents for B . cinerea includes bacteria within the genera of Bacillus and Pseudomonas , filamentous fungi within the genera of Ulocladium , Gliocladium and Trichoderma and also within the genera of Pichi a and Candida of yeast (Jacometti et al., 2010; Paulitz and Belanger, 2001) . Bacillus species including B. subtil is , B. a myloliquefaciens and B. mycoides have various modes of action including competition, parasitism, antibiosis, and induction of systemic acquired resistance (Choudhary and Johri, 2009; Pal and Gardener, 20 06; Paulit z and Belanger, 2001) . The mode of action of Trichoderma harzianum includes mycoparasitism, competition for nutrients or space and inactivation of enzymes produced by pathogens ( Vidhyasekaran , 2004). Similarly, Gliocladiu m catenulatum offers ant agonistic activity through antibiosis and Ulocladium oudemansi , Aureobasidium pullulans competes with B. cinerea for nutrition (Castoria et al., 2001; Jacometti et al., 2010; Pal and Gardener, 2006) Applying Gliocladiu m catenulatum (Prestop R WP) on geranium limited Botrytis blight in the greenhouse and significantly reduced disease incidence and severity ( Elmhirst et al., 2011) . This biological control agent also limited Botrytis stem canker on greenhouse tomatoe s (Utkhede and Mathur, 2006) . Tricho derma harzianum ( RootShield ) and Rhodosporidium diobavatum S33 strain sprayed as curati ve treatment on the wounded surface of greenhouse tomato stems reduced 10 lesion expansion caused by B . cinerea increasing yield (Utkhede and Mathur, 200 2) . Elad ( 1994) re ported the application of T . harzianum (0.5 - 1.0 g/l) in the vineyards significantly reduced grape gray mold disease incidence up to 78%. The T. hamatum 382 (T382) isolate suppressed Botrytis blight severity in begonia and geranium when applied as an amend ed form in the potting mix (Horst et al., 2005; Olson and Benson, 2007) . Binucleate Rhozoctonia (BNR) applied in the potting mix before transplanting also induced systemic resistance and re sulted in a reduction of disease symptoms on the geranium foliage (Olson and Benson, 2007) . Ingram and Meister (2006) found that Bacillus subtilis (Serenade ASO) and extract of Reynoutria sachalinensis (Milsana ) significantly reduced grey mold disease s everity of greenhouse tomatoes. Application of Ulocladium atrum on geranium stock plants decrease d B . cinerea conidial production with reduced severity on necrotic leaves (Gerlagh et al., 2001) . Bacillus velezensis ( strains 5YN8 and DSN012) suppressed growth and conidial formation of B. cinerea on pepper through the s ecreti on of secondary metabolites and releas e of volatile organic compounds ( Jiang et al., 2018) . M ycelial growth of B . cinerea was inhibited by Azotobacter chroococcu m . S porulation and severity on strawberry was reduced when Chlorella vulgaris was sprayed (El - ghanam et. al., 2015) . Aureobasidium pullulans, Gliocladium catenul atum and Chaetomium globosum reduced the Botrytis disease incidence and sporulation by 75% on stems of tomato and cucumber. Both A. pullulans and G. roseum completely prevented the disease on cucumbers grown in the greenhouse (Dik et. al., 1999) . In cyclamen, applications of U. atrum and G. roseum decreased disease incidence with a reduced number of petioles becoming infected in the greenhouse (Köhl et al., 1998) . Under highly conducive environmental conditions, T. hamatum 382 and binucleate Rhozoctonia (BNR) did not effectively control the B . cinerea on geranium. However, under a 11 less conducive environment the y reduced the disease severity as effectively as chemical fungicides (Olson and Benson, 2007) . CHEMICAL FUNGICIDES Complete h ost resistance to B. cinerea has not been identified fo r greenhous e ornamentals so fungicides are important (Yourman and Jeffers, 1999) . The fungicides used to control of B. cinerea include: (i) the benzimidazole fungicides ( carbendazim , benomyl , thiophanate methyl ) with anti - microbial properties ; (ii) phenylpyrrole fungicide ( fludioxonil ) and dicarboximide fungicide ( iprodione ) affecting fungal content of polyols, probably involved in osmoregulation; (i ii ) anilinopyrimidine fungicides ( cyprodinil and pyrimethanil ), a methionine biosynthesis inhibitor whose toxicity ability is reversed by amino acids; (iv) strobilurins (Quinone outside inhibitors or QoIs) fungicides (pyraclostrobin) being inhibitor of mitochondrial electron transport complex III and bin ding Qo site of cytochrome b ; (v) phenylpyridinamine fungicide ( fluazinam ) and Succcinate dehydrogenase (SDH I ) fungicid e ( boscalid ) a toxicants affecting fungal respiration ; and (v i ) hydroxyanilide fungicide ( fenhexamid ) a sterol biosynthesis inhibitor ( Leroux, 2007; Bardas et al., 2010; Hahn, 2014) . In addition to these site - specific fungicides representing different mode of action, multisite inhibitors (dithiocarbamates, captan, chlorothalonil ) have been used widely for a long period of time (Hahn, 2014) . Two fungicide groups; benzimidazole and dicarbox imides were initially highly effective against B. cinerea and were used intensively over decades (Elad and Shtienberg, 1995) . According to a study regarding the efficacy of six different classes of fungicides by Kim et al. (2016), the phenylpyrrole fungicide (fludioxonil) was most effective in inhibiti ng mycelial growth, germination and conidiation (EC50 < 0.1µg/ml). Boscalid, tebuconazole, iprodione and fenpyrazamine hav e an EC50 in the range of 0.3 to 0.9 µg/ml. Pyrimethanil has an EC50 of 50 1µg/ml and is less 12 effective in inhibiting mycelial growt h compared to fludioxonil. (Kim et al., 2016) . Mixing azoxystrobin with carbendazim or iprodione or applyin g azoxystrobin in alternation with carbendazim and iprodione were not effective in limiting fungicide resistance in a B . cinerea popu lation indicating multiple - resistance to different families of fungicides (Jiang et al. , 2009) . Using chemical fungicides wit h the same mode of action for a longer period to control Botrytis may result in pathogen resi stance (Gerlagh et al., 2001) . FUNGI CIDE RESISTANCE R esistance to fungicides has developed among B. cinere a isolates (Kim et al., 2016) . R esistance to benzimidazole ( thiophanate - methyl ) and dicarboximide ( vinclozolin ) was frequently detected in populations of B . cinerea in greenhouse - grown ornamentals (Yourman and Jeffers, 1999) . I solates resistan t to the benzimidazole fungicide ( benomyl ) was detected in all greenhouse and double resistance to both benzimidazole and dicarboximide was detected in six greenhouses ( Moorman and Lease , 1992). Yourman and Jeffers (1999) found that B. cinerea isol ates resista nt to dicarboximide were also resistant to benzimidazoles even though there had not been exposure to benzimidazole previously n or ha d the products been used for a long period. N egative - cross resistance has been reported between benzimidazoles ( e.g. carbend azim) and phenylcarbamates (e.g. diethofencarb) (Leroux, 2007) . Fungici de resistanc e may result from excessive use of fungicides with same mode of action , stability of fungicide - resistant isolates , or movement of resistant isolates via plant material while shipping from propagation to production greenhouses during various stages of plant growth. (Moorman and Lease, 1992; Yourman and Jeffers, 1999) . 13 Cross resistance to dicarboximide and benzimidazole fungicides was found among isolates accounting for 65.8% of the total (Moyano et al., 2004). Resistance to three fungicide clas ses including dicarboximides ( procymidone ), benzimidazole ( carbendazim ) an d N - phenylcarbamates ( diethofencarb ) was found in 14% of the isolates. Resistance to these three fungicide classes and anilinopyrimidines (pyrimethanil ) was found in 3% of the isola tes collected from commercial greenhouses of vegetable crops (cucumber, be an, tomato, squash, eggplant and pepper) in Spain (Moyano et al., 2004) . Isolates collected from orchards tre ated with a pyraclostrobin and boscalid mixture were resistant to both wit h an EC50 value greater than 50mg/l for boscalid and 16 to >50 mg/l for pyraclostrobin; none were resistant to fludioxonil or fenhexamid (Bardas et al., 2010; Markoglou et al. , 2006) . Cross resis tance studies showed that the mutation for pyraclostrobin resistance can reduce the sensitivity of mutant strains to other QoIs including azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin (Markoglou et al., 2006) . CULTIVAR RESISTANCE Screening different cultivars of ornamental crops in the greenhouse ha s shown some partial resistance against B . cinerea . Uch neat et al. (1999a) found different levels of resistance when evaluating forty - five g enotypes of Pelargonium against B. cinerea infection; two genotypes were consistently more resistant as measured by foliar lesion diameter. Uchneat et al. ( 1999 b ) studied floral infection to B. cinerea using sixty - two genotypes of Pelar gonium species and found varying level of resistance with diploid genotypes having greater resistance than tetraploid. Also, P. peltatum cultivars were found more resistant than P. x hortorum with regards to floral infection and no correlation was observed between floral and foliar resistance (Uchneat et al., 1999 b ) . Tian et al., ( 2019) screened 15 tree peonies for resistance to B. cinerea a nd found 14 different resistance levels with early flowering cultivars more resistant than late flowering cultivars. Krahl a nd Randle ( 1999) evaluated forty - eight petunia cultivars for resistance to B . cinerea and found a range of variation among cultivars over two seasons in the greenhouse; only one culti var was consistently resistant. Also, inconsistencies among cultivars re garding resistance were observed when different methods of inoculation were used for screening. Similarly, fluctuations in ranking lisianthus cultivars were observed by Wegulo and Vilchez ( 2007 ) when comparing different inoculation methods for resistance against B . cinerea. Selected ornamental crops were favored by B . cinerea infection as measured by the proportion infection: geranium plants were more susce ptible than petunia and impatiens under similar environment al conditions ( Prichard et.al, 1999). Lisianthus plants were more susceptible than rose and gerbera (Vrind, 2005) . Plant resistan ce to B. cinerea depends on the rate of senescence, structural defense and defenses accelerated by the production of different hormones (Elad and Evensen, 1995) . In biotrophs hypersensitive response is considered a major component for host resistance whereas the necrotrophic patho gens such as B. cinerea trigger a hypersensitive response for its pathogenicity. Hy persensitive response e nhanced generation of reactive oxygen species which facilitates its colonization and increases pathogenicity by using the host defense mechanism (Govrin and Levine, 2000) . Disease control for B. cinerea is difficult due to the ability of the pathogen to attack any plant growth stage. Senescent plant parts are easily invaded by the pathogen so the changes related with senescence can play a role in host resistance (Elad and Evensen, 1995) . Defense mechanism s against B . cinerea may be mediated by jasmonic acid, salicylic acid, abscisic acid and eth ylene signaling pathways; and are linked among a complex network (AbuQamar et al., 2017) . Likewise, nitric oxide was found to have an important role in 15 host resistance of geranium against B . cinerea with early nitric oxides bursts and production of seconda ry nitric oxide stimulating noncell - death - as sociated defense (Floryszak - wieczorek et. al, 2007) . Host resistance is important due to the development of fungicide resistance by key pathogens (Elad and Evensen, 1995) . Transgenic geranium plants with antimicrobi al protein Ace - AMP1 have increased resistance to B. cinerea based on sporulation density. There was a significant negative correlation between disease incidence and protein level signaling the inhibitory effect o f protein on disease development (Bi et al., 1999) . Similarly, plants expressing mannitol dehydro genase (MTD) protein also exhibited defense against B. cinerea although mannitol may act as a pathogenicity factor. Williamson et al. (2013 ) assessed effects of MTD expression on zonal geranium and showed that plants with overexpression of MTD have higher resistance to B. cinerea. So, Botrytis blight is one of the most important disease of greenhouse ornamentals causing higher economic loss to the growers. Chemical fungicides have been used in tensively to control this disease and the pathogen has developed resistance against the different classe s of fungicides. Growers were interested on the alternative management options that helps to better control of disease and avoid the fungicide resistance. Exploring the host resistance of the greenhouse ornamentals and making choice of the resistance culti var could help the growers to better design their management strategies. Also, the use of biorational products could be next alternatives for the control of Botrytis blight. The better understanding of the host resistance of cultivars and coupling with the biorational products could provide the grow ers with the good option for the management of Botrytis blight on the greenhouse ornamentals. 16 LITERATURE CITED 17 LITERATURE CITED 1. AbuQamar, S., Moustafa, K., and Tran, L. S. P. 2017. Mechanisms and strategies of plant defense against Botr ytis cinerea . Crit. Rev. Biotechnol . 37 : 262 274 . 2. Ahmed, A., Zaman, S., Mazid, M., Rahman, M., Sarkar, M., Arbia, L., Ud - deen, M., and Kabir, G. 2016. Studies of Botrytis cinerea causing botrytis gray mold disease in chickpea ( Cicer arietinum L.). J . Bio s c i . 22 : 69 - 76 . 3. Alderman, S. C., and Lacy, M. L. 198 4 . Influence of temperature and water potential on growth of Botrytis allii . Can . J . Bot . 62:1567 - 1570 . 4. Bardas, G. A., Veloukas, T., Koutita, O., and Karaoglanidis, G. S. 2010. Multiple resistance of Botry tis cinerea from kiwifruit to SDHIs, QoIs and fungi cides of other chemical groups. Pest Manag . Sci . 66 : 967 973. 5. Bi, Y. M., Cammue, B. P. A., Goodwin, P. H., KrishnaRaj, S., and Saxena, P. K. 1999. Resistance to Botrytis cinerea in scented geranium transf ormed with a gene encoding the antimicrobial protei n Ace - AMP1. Plant Cell Rep . 18: 835 840. 6. Castoria, R., De Curtis, F., Lima, G., Caputo, L., Pacifico, S., and De Cicco, V. 2001. Aureobasidium pullulans (LS - 30) an antagonist of postharvest pathogens of f ruits: Study on its modes of action. Postharvest Biol . and Technol. 22:7 17. 7. Choudhary, D. K., and Johri, B. N. 2009. Interactions of Bacillus spp . and plants - With special reference to induced systemi c resistance (ISR). Microbiol . Res. 164:493 513. 8. C oley - Smith, J. 1980. Sclerotia and other structures in survival . Pages 85 114 I n : The Biology of Botrytis . J. R. Coley - Smith , K. Verhoeff , and W. R. Jarvis , e ds. Academic Press, London, UK. 9. Daughtrey, M . L., and Benson, D. M. 2005. Principles of p lant h e alth m anagement for o rnamental p lants. Annu . Rev . Phytopathol . 43 : 141 169. 10. Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond - Kosack, K. E., Di Pietro, A., Spanu , P.D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J., and Foster, G.D. 2012. The top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 134 : 414 430 . 11. Dik, A. J., Koning, G., and Köhl, J. 1999. Evaluation of microbial antagonists for b iological control of Botrytis cinerea stem infection in cucumber and tomato. Eur . J . Plan t Pathol. 105 : 115 122. 18 12. Elad, Y., and Shtienberg, D. 1995. Botrytis cinerea in greenhouse vegetables: chemical, cultural, physiological, and biological controls and th eir integration. Integr . Pest Manag . Rev. 1:15 29. 13. Elad, Ygal, and Evensen, K. 1995. P hy siological aspcects of resistance to Botrytis cinerea . Phytopathology. 85:637 - 643. 14. Elad , Yigal. 1994. Biological control of grape grey mould by Trichoderma harzianum . Crop Prot . 13 : 35 38. 15. Elad, Yigal, Williamson, B., Tudzynski, P., and Delen, N. 2007. B otrytis Spp and disease they cause in agricultural system - A n introduction. Pages 1 - 8. In: Botrytis : Biology, Pathology and Control . Y. Elad, B. Williamson, P. Tudzynski and N. Delen. ( eds. ) Kluwer Academic P ublishers. Dordrecht, The Netherlands . 16. El - ghana m A . A, Farfour S . A . , and Ragab , S . S . 2015 . Bio - suppression of strawberry fruit rot disease caused by Botrytis cinerea . J . Plant Pathol . Microbiol . S3:005 . 17. Elmhirst, J. F., Haselhan, C., and Punja, Z. K. 2011. Evaluation of biological control agents for control of botrytis blight of geranium and powdery mildew of rose. Ca n. J . Plant Pathol. 33:499 505. 18. Floryszak - wieczorek, J., Arasimowicz, M., Milczarek, G., Jelen, H., and Jackowiak, H. 2007. Only an early nitric oxide burst and the following wave of se condary nitric oxide generation enhanced effective defense responses of Pelargonium to a necrotrophic pathogen, New Phytol. 175: 718 730. 19. Gerlagh, M., Amsing, J. J., Molhoek, W. M. L., Bosker - Van Zessen, A. I., Lombaers - Van Der Plas, C. H., and Köhl, J. 20 01. The effect of treatment with Ulocladium atrum on Botrytis cinerea - attack of geranium ( Pelargonium zonale ) stock plants and cuttings. Eu r. J . Plant Pathol. 107:377 386. 20. Govrin, E. M., and Levine, A. 2000. The hypersensitive response facilitates plant i nfection by the necotrophic pathogen Botrytis cinerea . Curr . Biol . 10 : 751 757. 21. Hahn, M. 2014. The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study. J . Chem . Biol . 7 : 133 141. 22. Hausbeck, M . K., and Moorman, G. W. 199 6. Managing Botrytis in greenhouse - grown flower Crops. Plant Dis. 80:1212 - 1219. 23. Hausbeck, M. K., and Pennypacker, S. P. 1991a. Influence of g rower a ctivity and d isease i ncidence on c oncentrations of a irborne c onidia of Brotytis cinerea a mong g eranium stoc k plants. Plant Dis . 75 : 798 803. 19 24. Hausbeck, M. K., and Pennypacker, S. P. 1991b. Influence of grower activity on concentrations of airborne conidia of Botrytis cinerea among geranium cuttings. Plant Dis . 75 : 1236 1243. 25. Hausbeck, M., Pennypacker, S., and Ste venson, R. 1996. The effect of plastic mulch and forced heated air on Botrytis cinerea on geranium stock plants in a research greenhouse. Plant Dis. 80:170 - 173. 26. Holz, G., Coertze, S., and Williamson, B. 2007. The ecology of Botrytis on plant surfaces. Pag es 9 - 27 In: Botrytis : Biology, Pathology and Control. Y. Elad, B. Williamson, P. Tudzynski and N. Delen. ( eds. ) Kluwer Academic Publishers. Dordrecht, The Netherlands. 27. Horst, L. E., Locke, J., Krause, C. R., McMahon, R. W., Madden, L. V., and Hoitink, H. A. J. 2005. Suppression of Botrytis blight of begonia by Trichoderma hamatum 382 in peat and compost - amended po tting mixes. Plant Dis. 89:1195 1200. 28. Ingram, D. M., and Meister, C. W. 2006. Managing Botrytis gray mold in g reenhouse tomatoes using tradition al and bio - fungicides. Plant Health Prog . 7 : 1 7. 29. Jacometti , M. A., Wratten, S. D., and Walter, M. 2010. Review: Alternatives to synthetic fungicides for Botrytis cinerea management in vineyards. Aust . J . Grape Wine R . 16:154 172. 30. Jarvis, W. R. 1960. An apparatus for studying hygroscopic responses in fungal conidiophores. Trans . Brit . Mycol . Soc . 43 : 525 - 52 8. 31. Jarvis, W. R. 1989. Managing d iseases in g reenhouse c rops. Plant Disease. 73(3):190 - 194. 32. Jiang, C., Liao, M., Wang, H., Zheng, M., and Xu, J. 2018. Bacillus velezensis , a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea . Biol . Control. 126:147 157. 33. Jiang, J., Ding, L., Michailides, T. J., Li, H., and Ma, Z. 2009. Molecular characterization of field a zoxystrobin - resistant isolates of Botrytis cinerea . Pestic . Biochem . Phys . 93 : 72 76. 34. Kim, J. - O., Shin, J. - H., Gumilang, A., Chung, K., Choi, K. Y., and Kim, K. S. 2016. E ffectiveness of different classes of fungicides on Botrytis cinerea causing gray mol d on fruit and vegetables. Plant P athol . J . 32 : 570 574. 20 35. Köhl, J., Gerlagh, M., De Haas, B. H., and Krijger, M. C. 1998. Biological control of Botrytis cinerea in cyclamen with Ulocladium atrum and Gliocladium roseum under commercial growing conditions. Phy topathology . 88 : 568 575. 36. Krahl, K . H., and Randle, W. M. 1999. Resistance of petunia phenotypes to Botrytis cinerea . HortScience. 34:690 692. 37. Kritzman, G., and Netzer , D. 1978. A selective medium for isolation and identification of Botrytis spp. from s oi l and onion s eed. Phytoparasitica . 6 : 3 7. 38. Leroux, P. 2007. Chemical control of botrytis and its resistance to chemical fungicides. Pages 195 - 222 . In: Botrytis : Biology , Pathology and Control. Y. Elad, B. Williamson, P. Tudzynski and N. Delen. ( eds ) . Kluw er Academic P ublishers. Dordrecht, The Netherlands . 39. Louis, C., Girard, M., Kuhl, G., and Lopez - Ferber, M. 1996. Persistence of Botrytis cinerea in its vector Drosophil a melanogaster . Phytopathology. 86:934 - 939. 40. Maas, J.L. (1969) E ff ect of time and temper ature of storage on viability of Botrytis convoluta conidia and sclerotia. Plant Dis ease Reporter . 53 : 141 - 144. 41. Markoglou, A. N., Malandrakis, A. A., Vitoratos , A. G., and Ziogas, B. N. 2006. Characterization of laboratory mutants of Botrytis cinerea resis tant to QoI fungicides. Eur . J . Plant Pathol . 115 : 149 162. 42. Moorman, G. W., and Lease, R. J. 1992. Benzimidazole and d icarboximide r esistant Botrytis cinerea from Pennsylvania g reenhouses. Plant Dis . 76 : 477 480. 43. Moyano, C., Gómez, V., and Melgarejo, P. 2 004. Resistance to pyrimethanil and other fungicides in Botrytis cinerea populations collected on vegetable crops in Spain. J . Phytopathol . 152 : 484 490. 44. Olson, H. A., and Benson, D. M. 2007. Induced systemic resistance and the role of binucleate Rhizocto nia and Trichoderma hamatum 382 in biocontrol of Botrytis blight in geranium. Biol . Control. 42:233 - 241. 45. Pal, K. K., and Gardener, B. M. 2006. Biological control of plant pathogens. The Plant Health Instructor. 2:1117 - 1142. 46. Pande, S., Singh, G., Narayana Rao, J., Bakr, M. A., Chaurasia, P. C. P., Joshi, S., Johansen, C., Singh, S. D., Kumar, J., Rahman, M.M., and Gowda, C. L. L. 200 1 . Integrated management of Botrytis gray mold of chickpea. Information Bulletin no. 61. ICRISAT, Patancheru, Andhra Pradesh, India. 21 47. Paulitz, T. C., and Belanger, R. R. 2001. Biological control in greenhouse system. Annu . Rev . Phytopathol. 39:103 133. 48. Pritchard , P . M. 1995. Influence of DIF on the susceptibility of floral crops to Botrytis cinerea . MS thesis. Mic higan State Uni v ersity . E ast Lansing. 49. Pun ja, Z. K., and Utkhede, R. S. 2003. Using fungi and yeasts to manage vegetable crop diseases. Trends Biotechnol. 21:400 407. 50. Salinas, J., Glandorf, D. C. M., Picavet, F. D., and Verhoeff, K. 1989. Effects of temperature, relativ e humidity and age of conid ia on the incidence of spotting on gerbera flowers caused by Botrytis cinerea . Neth . J . Plant Pathol . 95 : 51 64. 51. Shiraishi, M., Fukutomi, M. and Akai, S. 1970 a. Effect of temperature on the conidium germination and appressorium f ormation of Botrytis cinerea Pers. Ann . Phytopathol . Soc . Japan . 36 : 234 - 23 6. 52. Shiraishi, M., Fukutomi, M., and Akai, S. 1970 b . On the mycelial growth and sporulation of Botrytis cinerea Pers. and the conidium germination and appressorium formation as affec ted by the c onidial age. Ann . Phytopathol . Soc . Japan . 36:230 - 233. 53. Sirjusingh, C., and Sutton, J. C. 1996. Effects of wetness duration and temperature on infection of geranium by Botrytis cinerea . Plant Dis . 80 : 160 165. 54. Sirjusingh, C., Sutton, J. C., and Tsujita, M. J. 1996. Effects of inoculum concentration and host age on infection of g e ranium by Botrytis cinerea . Plant Dis. 80:154 - 159. 55. Steiger, D. 2007. Global ecoonomic importance of Botrytis protection. Page 7. In : 14 th I nternational B otrytis S ymposi um. African Sun MeDIA Pty (Ltd.). 56. Tian, Y., Che, Z., Sun, D., Yang, Y., Lin, X., and L iu, S. 2019. Resistance identification of tree peony cultivars of different flowering time to gray mold pathogen Botrytis cinerea . HortScience . 54 : 328 330. 57. Uchneat, M. S., Spicer, K., and Craig, R. 1999a. Differential response to floral infection by Botrytis cinerea within the genus Pelargonium . HortScience. 34:718 - 720. 58. Uchneat, M. S., Zhigilei, A., and Craig, R. 1999b. Differential r esponse to f oliar i nfection with Bot rytis cinerea withi n the g enus Pelargonium . J . Am . Soc . Hort ic. Sci . 124:76 80. 59. USDA - National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. Retrieved from: https://www.nass.usda.gov/Publications/Todays_Reports/reports/floran19.pd f 22 60. Utkhede, R. S., a nd Mathur, S. 2002. Biological control of stem canker of greenhouse tomatoes caused by Botrytis cinerea . Can . J . Microbiol . 48 : 550 554. 61. Utkhede, R. S., and Mathur, S. 2006. Preventive and curative biological treatments for control of Bo trytis cinerea stem canker of greenhouse tomatoes. Bio l. Control . 51:363 373. 62. Van Den Berg, L., and Lentz, C. P. 1968. The effect of relative humidity and temperature on surv ival and growth of Botrytis cinerea and Sclerotinia sclerotiorum . Can . J . Bot . 46 : 1477 1481. 63. Vidhyasekaran, P. 2004. Biological control Microbial pesticides. Pages 239 270 . In: Concise encyclopedia of plant pathology. Food Products Press. Binghamton, US A. 64. Vrind, T. A. 2005. The Botrytis problem in figures. Acta Hortic . 669 : 99 102. 65. Wegulo, S. N., and Vilchez, M. 2007. Evaluation of l isianthus c ultivars for r esistance to Botrytis cinerea . Plant Dis . 91:997 1001. 66. Whetzel , H. H. 1945. A synopsis of the genera and species of the sclerotiniaceae, a family of stromatic inoperculate dis comycetes. Mycologia . 37 : 648 - 714 . 67. Williamson, B., Tudzynski, B., Tudzynski, P., and Van Kan, J. A. L. 2007. Botrytis cinerea : The cause of grey mould disease. Mol . Plant Pathol. 8:561 580. 68. Williamson, J. D., Desai, A., Krasnyanski, S. F., Ding, F., Guo, W., Nguyen, T., Olson, H. A., Dole, J. M., and Allen, G. C. 2013. Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea . Plant Cell Tiss . Org. 115: 367 375. 69. Yourm an, L. F., and Jeffers, S. N. 1999. Resistance to b enzimidazole and d icarboximide f ungicides in g reenhouse i solates of Botrytis cinerea . Plant Dis . 83:569 575. 23 CHAPTER 1. EVALUATION OF GERANIUM CULTIVARS AND BIORATIONAL PRODUCTS TO CONTROL BOTRYTIS BLIGHT IN GREENHOUSE 24 ABSTRACT Botrytis blight caused by the fungus Botrytis cinerea is one of the most important disease of greenhouse - grown ornamental crops. On geranium, i t causes leaf, stem, and flower blight and decreases its marketability . Our objectives were to evaluate (i) susceptibility of geranium cultivars to B. cinerea and (ii ) efficacy of different biorational products for control of Botrytis blight on geranium. Disease assessment included the number of blighted leaves, foliar lesio ns, and leaves with B. cinerea sporulati on. A rea under disease progress curve (AUDPC) was calculated to determine overall disease progress. Among the ten g eranium cultivars evaluated, re significantly more resistant than for all measured parameters and AUDPC data. When ten treatments were compared in the efficacy trial of biorational products, Aureobasidium pullulans (Botector) and Gli ocladium catenulatum (Prestop) effectively co ntrol ed the disease according to AUDPC for blighted leaves and leaves with sporulating Botrytis in both Orange . Pseudomonas chlororaphis (Zio) , B acillus amyloliquefaciens (Serifel) , B . subtilis ( Serenade Opti ) and B. mycoides ( LifeGard ) were also effective in the moderately resistant geranium based on AUDPC values for all measured parameters. AUDPC for leaves with s porulating B . cinerea showed that al l biorational products included in the study effective ly control led B . cinerea e xcept Streptomyces lydicus ( Actinovate ) in geranium . The moderately resistant geranium cultivars could be used in combination with biorational controls f or effective Botrytis b light control for a more sustainable management approach. 25 INTRODUCTION The floricultural industry is an important contributor to U.S. agriculture producing $4.63 billion in revenue in 2018 ( USDA - N ASS , 2019) . Geraniums are a popular flowering annual with a yearly revenue of $119.8 million which includes seeded flats ($7.6 million) , hanging baskets ( $ 30.5 million ) and pots of cutting - propagated geranium s ( $81.6 million) ( USDA - N ASS , 2019) . Geranium is susceptible to the plant pathogen Botrytis cinerea (teleomorph: Botryotinia fuckeliana ), an airborne necrotrop h ic fung us, considere d to be one of the most common and destructive pathogens ( Dean et al. , 2012 ; Chandel and Kumar, 2018) . Initial symptoms include water - so ak ed tissue, brown spot ting, and soft rot resulting in blossom blight, leaf blight, bud rot, stem and crown rot , stem can k er , and damping - off (H ausbec k and Moorman, 1996; Jiang et al., 2018; Williamson et al., 2007) . The grey conidial masses that form on infected tissue are diagnostic and are commonly called grey mold (Pu nja and Utkhede, 2003; Williamson et al., 2007) . The optimum temperature for conidial germination is 22 to 25 0 C although g ermination of conidia and lesion development may occur between 4 and 25 0 C (Salina s et al. , 1989) . T he optimum temperature for infection is 15 to 25 0 C. (Jarvis, 1989) W et and humid greenhouse conditions especially during propagation promote conidial germina tion and coalescence of lesions which reduces plant quality (Hausbeck and Pennypacker, 1991) . I nfe ction may be intitated on senescing flowers or leaves near the moist soil surface (Hausbeck and Harlan, 2020) . H ost resistance against B. cinerea in greenhouse ornamental s has not been identified (Yourman a n d Jeffers, 1999) although quantitative resistance to B . cinerea was observed among different genotypes of geranium by Uchneat et al. ( 1999 b ) . Currently , s uccessful limitation of B . cinerea requires an integrated approa ch including sanitation, environmental manipulation such as heating and venting, and timely application of effective fungicides (Jarvis, 1989 ; Hausbeck and 26 Moorma n, 1996 ) . The greenhouse en vironment including warm temperatures, high relative humidity, periods of leaf wetness, and limited air exchange favor Botrytis blight (Elad and Shtienberg, 1995; Paulitz and Belanger, 2001) . Reliance on chemical fungicides with the same mode of ac tion may result in pathogen re sistance (Gerlagh et al., 2001) . R esistance to fungicides has developed among B. cinere a isolates ( Hahn, 2014; Ki m et al., 2016) . R esistance to benzimidazole ( thiophanate - methyl ) and dicarboximide ( vinclozolin ) were frequently detected in populations of B . cinerea in greenhouse - grown ornamentals (Yourman an d Jeffers, 1999) . Multiple fungicide resistance a mong B. cinerea isolates to various chemical classes with different modes of action has been reported on cut rose s (Muñoz et al., 2019) and petunia (Samarakoon et al., 2017). Fungicide resistant B . cinerea isolates may be disseminated via plant material that is shipped from propagation to production greenhouses (Moorman and Lease, 1992; Yourman and Jeffers, 1999) . Biorational products offer an alternative to chemical fungicides . S uppression of B . cinerea has been reported from products containing species of Bacillus , Pseudomona s , Ulocladium , Gliocladium , Trichoderma , Pichia or Candida (Jacometti et al., 2010; Paulitz and Belanger, 2001) . There are various mechanism s by which living organisms may suppress B. cinerea including induction of systemic acquired resistance (Choudhary and Johri, 2009 ) , competition for nutri ents , myco parasitism and antibiosis (Pal and Gardener, 2006; Paulitz and Belanger, 2001 ; Vidhyasekaran, 2004) . F oliar sprays of Gliocladiu m catenulatum reduced Botrytis blight incidence and severity on geranium (Elmhirst et al. , 2011) . Applica tion of Ulocladium atrum to geranium stock plants decrease d the percentage of necrotic leaves and pathogen sporulation (Gerlagh et al., 2001) . T richoderma hamatum ( isolate 382) suppress ed Botrytis blight severity in begonia and geranium when incorporat ed in to the potting m edium 27 (Horst et al., 2005; Olson and Benson, 2007) . Binucleate Rh i zoctonia (BNR) isolates BNR621 and P9023 a dded to the potting mix prior to transplanting induce d systemic resistance and reduc ed foliar disease on geranium (Olson and Benson, 2007) . H ost resistance combined with birational products would provide a sustainable disease approach. Our objectives were to: 1) Identify resistance to B . cinerea among selected geranium cultivars and 2 ) Evaluate the efficacy of biorational products i n limit ing Botrytis blight. MATERIALS AND METHODS I noculation and incubation . A B. cinerea isolate w as obtained from symptomatic geraniums growing in the Plant Science G reenhouse s at Michigan State Univers ity (MSU) , East Lansing, MI . Hyphae from symptomatic foliage was teased out of the tissue using a needle and placed onto potato dextrose agar media (PDA) (39 g PDA, 1000 ml H 2 0 ) in 10 - cm diameter petri plates and grown for 14 days at 20 - 25 0 C under continuo us fluorescent light to prompt sporulat ion . Single spore cultures from this isolate w ere obtained by transferring conidia o nto water agar media ( 16g agar, 1000 ml H 2 0 ) and plac ing them under the fluorescent light for a pproximately one week. A single hypha was selected from the water - agar medi a using a stereo light microscope and transferred to another culture plate containing PDA to establish a pure colony which was then stored in silica gel in the refrigerator at - 4 0 C. I ron baskets (n=100) were bleached ( S odium hypochlorite (0.65%), C lorox germicidal bleach, The Cloro x company, Oakland, CA) and placed in translucent plastic bags (21 x 5.5 x 38 cm 3 ) containing enough water to cover the bottom of basket for increased relative humidity (RH) . P lants were select ed and placed inside the iron basket and arranged in completely randomized design on a bench in the Plant Science G reenhouse s at MSU that was shaded ( 80% ) . 28 To inoculat e the geraniums, a conidial suspension was pre pared by flooding 11 - day - old B. cinerea cul ture s grown on PDA with sterilized distilled water and scraping with a spatula to dislodge the conidia . The conidial suspension was strained through cheesecloth and standardized to 1 x 10 6 conidia/ml solution usin g a hemocytometer. O n 26 Oct , the B. cinere a conidial suspension was spray ed on each plant with a hand sprayer until run off. Each plant w as enclosed in a translucent plastic bag containing water at the bottom that was sealed to provide high RH for incubat ion . The plants remained in the bags for th e entire duration of the experiment. A Watchdog A - series data logger (Spectrum technologies Inc., Aurora, IL) was placed in one bag to record temperature and RH at hourly intervals. Cultivar e valuation . Ten g eranium cultivars ( Pelargonium x hortorum and P. peltatum ) representing different colors were chosen (Ball Horticultural Company, West Chicago, IL) (Table 1). Seed w as sown in 128 - cell plug trays containing soilless root medium (Suremix Perlite, Michigan Growers Products Inc, Galesburg, MI) on 16 Aug 20 18 and placed on a bench in the Plant Science G reenhouse s at MSU . S eedlings were transplanted on 1 Oct 2018 into square pots (10*10 cm 2 ) fill ed with soilless media and fertilized daily with 200 ppm water - soluble 20 - 20 - 20 water soluble NPK fertilizer (ICL S pecialty fertilizers, Dublin, OH) . Transplanted geraniums were drenched with the fungicide Subdue Maxx ( 0.08 ml/l, mefenoxam 22% , Syngenta Cr op Protection, LLC , Greensboro, NC ) to prevent root rot incited by Pythium spp. Average greenhouse air temperature during the growing period (16 Aug to Oct 26 ) was 23.6 0 C and the maximum/minimum temperatures were 33.5 0 C/13.5 0 C. Ten plants from each cultivar served as single plant replicat es. D isease was assess ed 7 days after the inoculation on 2 Nov 2018 with subsequen t assessments 13 (8 Nov) and 20 (15 Nov) days post inoculation. Average temperature was 18.4 0 C , rang ing from 12.2 0 C 21.9 0 C with RH of 89 to 100% inside 29 the plastic bags during incubation. The experiment was repeated following the same procedure with gera nium plants grown on the greenhouse (7 Mar to 17 May 2019). Plants were inoculated with Botrytis conidial su spension ( 17 May ) and incubated inside the plastic bag. D isease was assessed 7, 13 and 20 - days post inoculation on 24, 30 May and 6 Jun, respectivel y . Ma x imum /min imum temperature inside plastic bag during the incubation w ere 32.7 0 C/21.3 0 C with the average of 25 0 C and RH of 91 - 100%. Evaluation of bio rational products . geraniums determined to be moderately resistant and highly susceptible to Botrytis blight, respectively, were included . S eed s were planted into 128 - cell plug trays and grown in the Plant Science G reenhouse s of MSU for 45 days after which they were transplanted into square pots (10*10 cm 2 ) fill ed with soilless root medium and fertilized daily with 200 ppm 20 - 20 - 20 water soluble NPK fertilizer (ICL Specialty Fertilizers, Dublin, OH). Treatments included t en biorational products , a fungicide standard, and an untreated inoculated control (Table 2) . F ive and six , single - plant replications of and (n=72) , respectively, were included . Treatment s were applied three times with a hand compressed air sprayer at weekly interval s on 19, 26 Sep and 3 Oct 2019, except Gliocladium catenulatum (Prestop) wh ich was applied once due to the labeled application interval of 21 days. Plants were inoculated 1 day after the first treatment was applied on 20 Sep . Disease assessment was conducted on 26 Sep, and 3 and 10 Oct. Average temperature of 22.9 0 C was recorded during the incubation inside plastic bag with max./min. temperature of 30.8 0 C/18.9 0 C. The experiment was repeated following the same procedure from 17 Oct to 7 Nov 2019. Treatments were applied on 17, 24 and 31 Oc t at weekly interval. P lants were inoculated once with Botrytis conidial suspension (18 Oct), a day after the first application and i ncubated inside the plastic bag. Disease 30 assessment w as done on 6, 13 and 20 - days post inoculation on 24 , 31 Oct and 7 Nov, respectively. Maximum/minimum temperature inside the plastic bag s during the incubation were 21.4 0 C/ 16.4 0 C with the average of 2 1 0 C and RH of 9 2 - 100%. 31 Table 1. Geranium species and c ultivar s evaluated for susceptibility to Botrytis cinerea . Cultivars Species Pelargonium x hortorum P. x hortorum Ivy Tornado White P . peltatum P. x hortorum P. x hortorum P. x hortorum P. x hortorum P. x hortorum P. x hortorum P. x hortorum 32 Table 2 . Biorational products and a standard fungicide evaluated for efficacy against Botrytis cinerea on geranium. Products Active Ingredients Company Rate/ 100 gal App lication Interval (days) Actinovate® SP Streptomyces lydicus WYEC108 (0.037%) Novozymes BioAg Inc. 12 oz 7 Botector® Aureobasidium pullulans strain DSM 14940 (40%), DSM 14941 (40%) Bio - ferm 10 oz 7 Ulocladium oudemansii strain U 3 BioWorks , Inc. 4 lb 7 Extract of Swinglea glutinosa (82%) Gowan Company 2 pt 7 Bacillus mycoides (40%) Certis USA 4.5 oz 7 Prestop® WP Gliocladium catenulatum strain J1446 (32%) Danstar Ferment AG 70 oz 21 PureCrop1 Soybean oil (10%), Corn oil (5%) PureCrop1 200 oz 7 Serenade Opti® WP Bacillus subtilis QST713 (26.2%) Bayer CropScience Inc. 20 oz 7 Serifel® Bacillus amyloliquefaciens strain MBI600 (11%) BASF Corporation 16 oz 7 Pseudomonas chlororaphis strain AFS0 09 SePRO Corporation 100 oz 7 Decree® 50 WDG Fenhexamid (50%) SePro Corporation 1 lb 7 33 Disea se assessment and statistical analysis . The number of blighted leaves , foliar lesions and leaves with sporulating B . cinerea were counted. Area under the disea se progression curve (AUDPC) was calculated to express the cumulative disease on the geranium plants by using the formula AUDPC = i + y i+1 )/2] x (t i+1 t i ) where y i is the assessment of disease at i th observation, t i is the time (days) at the i th observation and n is the total number of observations ( Simko and Piepho, 2012 ). AUDPC was calculated for blighted leaves, foliar lesions and leaves with sporulating B . cinerea . Data w ere analyzed with a one - way ANOVA using PROC GLM procedure of SAS sta tistical analysis software ( SAS Institute Inc., Cary, NC, 2013 ) for the total number of blighted leaves, foliar lesions and leaves with the sporulating pathogen. The AUDPC of all the assessed parameters w as calculated based on three ratings. The assumption of the normality was satisfied in all of the trials which was checked using residual plots . The homogeneity of the varianc e biorational trials. Analysis of data showed unequal variance so the re - analysis was conducted using Sat terthwaite test for unequal variance for the adjustment in degree of freedom. LS Means were determined using PROC GLIMMIX procedure i n SAS and statistical differences between treatments within the trials w ere compared east S ignificant D i fference (LSD) t - test at the significance level of 0.05 (P = 0.05). 34 RESULTS Cultivar e valuation. of blighted leaves, foliar lesions and leaves with sporulating B. ci nerea for all assessed parameters in both trials and were significantly more resistant 00 (P<0.0001) (Table 2) (Trials 1 and 2) except for leaves with pathogen sporulation ( Trial 2 ) B . cinerea in both trials , bu Trial 2, A ccording to the AUDPC data for blighted leaves , foliar lesions and leaves with sporulating B . c inerea and (Trials 1 were more resistant than (Table C values for all parameters and were similar to parameters according to AUDPC data. 35 Table 3 . Mean number of blighted leaves , foliar lesions and leaves with sporulating B otrytis cinerea on geranium cultivars 20 days following inoculat ion . Cultivar s B lighted leave s (no.) Foliar lesions (no.) L eaves with B. cinerea sporulati on (no.) Trial 1 Trial 2 Tr ial 1 Trial 2 Trial 1 Trial 2 Ringo 2000 Violet 26.9 a 19.4 a 28.2 a 20.0 a 20.8 a 17.3 a x Maverick Scarlet Picotee 25.2 ab 18.1 a 27.1 ab 20.3 a 18.7 a - c 15.4 ab Multibloom Lavender 24.2 a - c 11.2 c 26.2 ab 12.0 c 19.6 ab 10.0 c Nano Deep Rose 23.0 a - d 9.1 c 23.1 a - c 9.1 c 16.6 a - d 8.4 c BullsEye Red 21.3 b - d 18.5 a 23.0 a - c 18.6 a 15.5 b - d 17.1 a Pinto Pink 19.9 b - e 17.1 ab 22.3 bc 17.4 ab 14.1 c - e 16.2 ab Quantum Salmon 19.1 c - e 12.4 c 19.9 cd 12.4 c 13.7 de 11.9 bc Horizon Coral Spice 18.0 d - f 11 .4 c 19.7 cd 12.3 c 12.8 d - f 9.9 c Pinto Premium Orange 15.2 ef 11.4 c 16.4 de 11.8 c 10.5 ef 10.2 c Ivy Tornado White 13.1 f 12.5 bc 13.6 e 12.9 bc 8.9 f 10.6 c x Column SD; P=0.05). 36 Table 4 . Area under disease progress curve (AUDPC) values for blighted leaves, foliar lesions and leaves with sporulating B otrytis cinerea on geranium cultivars when inoculated . Cultivars AUDPC for blighted leaves AUDPC for foliar lesi ons AUDPC for leaves with B. cinerea sporulation x Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Ringo 2000 Violet 186.20 a - c 141.40 ab 201.9 a - c 106.00 ab 124.65 a - c 106.00 ab y Maverick Scarlet Picotee 220.15 a 134.55 a - c 242.75 a 102.10 a - d 131.45 a b 102.10 a - d Multibloom Lavender 194.60 ab 87.40 de 209.50 ab 70.05 de 141.30 a 70.05 de Nano Deep Rose 181.70 a - c 54.40 e 184.65 b - e 44.55 e 122.35 a - c 44.55 e BullsEye Red 166.55 b - d 149.15 a 190.25 b - d 125.25 a 95.15 cd 125.25 a Pinto Pink 158.50 b - d 148.85 a 181.90 b - e 127.75 a 93.25 cd 127.75 a Quantum Salmon 149.35 cd 115.45 a - d 158.30 c - e 103.90 a - c 99.25 b - d 103.90 a - c Horizon Coral Spice 135.05 d 91.80 d 151.25 de 70.00 de 86.05 d 70.00 de Pinto Premium Orange 132.25 d 102.45 cd 147.45 d e 73.55 c - e 84.50 d 73.55 c - e Ivy Tornado White 129.60 d 111.50 b - d 136.65 e 85.15 b - d 82.65 d 85.15 b - d x Disease assessment were done on 7, 13 and 20 - days post inoculation on 2, 8 and 15 Nov 2018 (Trial 1) and 24, 30 May and 6 Jun 2019 (Trial 2). y Col 37 Figure 1 . Highly susceptible (A,B) and moderately resistant (C,D) g eranium cultivars observed 20 days after inoculation with Botrytis cinere a A: Ringo 2000 Violet, B: Maverick Scarlet Picottee, C: Pinto Premiun Orange, D: Horizon Coral Spice A B C D 38 Bi o rational evaluation on . Disease pressure was higher in Trial 1 than Trial 2. In Trial 2, Botrytis blight on the control plants wa s not advanced and was similar to the fungicide fenhexamid (Decree) fo r blighted leaves and foliar lesions (Table 4). However, according to the AUDPC data, plants treated with fenhexamid were less diseased than the control in both trials for all parameters . I n Trial 1, most of the biorational products provided control simila r to both the fungicide standard and the control according to the last observation and the AUDPC data. While applications of e xtract of Swinglea glutinosa ( Ecoswing ) resulted in protecti on similar to fenhexamid based on the last disease assessment (Trials 1 and 2) and the AUDPC values (Trial 2), AUDPC results from Trial 1 indicated that this biorational product was less effective than fenhexamid . Pseudomonas chlororaphis ( Zio ) (Trials 1 a nd 2) and Streptomyces lydicus ( Actinovate ) (Trial 2) w ere less effective than fenhexamid . 39 Table 5 . Mean n umber of blighted leaves , foliar lesions and leaves with sporulating B otrytis cinerea treated with biorational products and a fungicide standard 20 days following inoculation . Treatment (Trade name/active ingredient) Blighted leaves (no.) Foliar lesions (no.) Leaves with B. cinerea sporulation Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Fenhexamid (Decree) 2 5.2 c 4.50 d 25.5 d 4.7 d 22.5 b 2.3 d x Aureobasidium pullulans (Botector) 25.8 bc 13.0 bc 26.3 cd 13.0 bc 22.2 b 11.7 a - c Bacillus amyloliquefaciens (Serifel) 28.0 a - c 18.0 ab 28.0 b - d 18.5 ab 26.2 ab 15.3 a Gliocladium catenulatum (Prestop) 28.8 a - c 1 8.3 ab 29.8 a - d 18.3 ab 25.5 ab 15.5 a Bacillus mycoides (LifeGard) 28.8 a - c 13.2 bc 29.3 a - d 13.0 bc 26.3 ab 11.8 a - c Extract. Swinglea glutinosa (Ecoswing) 29.7 a - c 9.2 cd 30.3 a - d 9.2 cd 27.8 ab 6.7 cd Bacillus subtilis (Serenade Opti) 29.8 a - c 15.5 a - c 30.3 a - d 15.5 a - c 28.2 ab 12.5 ab Soybean and corn oil (PureCrop1) 31.3 a - c 15.5 a - c 32.3 a - d 15.5 a - c 29.5 ab 13.3 ab Ulocladium oudemansii (BotryStop) 32.5 a - c 19.7 a 33.7 a - c 19.7 a 28.0 ab 16.2 a Streptomyces lydicus (Actinovate) 33.5 ab 14.8 a - c 34.5 ab 14.8 a - c 30.5 a 12.7 ab Pseudomonas chlororaphis (Zio) 35.2 a 12.2 bc 35.5 ab 12.2 bc 31.3 a 10.5 a - c Untreated inoculated control 35.0 a 10.7 cd 36.2 a 10.7 cd 32.2 a 9.2 bc x Column means with a letter in common are not statistically differen P=0.05). 40 Table 6 . Area under disease progress curve (AUDPC) data for blighted leaves, foliar lesions and leaves with sporulating B otrytis cinerea treated with bio rational products . Treatment (Trade name/active ingredient) AUDPC for blighted l eaves AUDPC for foliar lesions AUDPC for l eaves with B. cinerea sporulati on x Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Fenhexamid (Decree) 191.92 c 35.58 d 207.67 d 39.67 d 125.42 c 13.42 c y Aureobasid ium pullulans ( Botector ) 215.83 bc 93.92 bc 224.00 cd 99.75 bc 157.50 bc 75.83 ab Bacillus amyloliquefaciens ( Serifel ) 214.08 bc 141.17 ab 224.58 cd 147.58 ab 172.67 a - c 117.83 a Gliocladium catenulatum ( Prestop ) 215.25 bc 135.33 ab 229.25 cd 138.83 ab 1 35.33 c 104.42 a Bacillus mycoides ( LifeGard ) 242.67 a - c 113.75 a - c 255.50 a - d 113.75 a - c 184.92 ab 95.08 a Extract. Swinglea glutinosa ( Ecoswing ) 258.42 ab 74.08 cd 282.33 a - c 75.25 cd 196.58 ab 50.75 bc Bacillus subtilis ( Serenade Opti ) 240.92 a - c 120 .75 a - c 250.25 b - d 127.17 a - c 172.08 a - c 96.25 a Soybean and corn oil ( PureCrop1 ) 274.17 ab 119.00 a - c 289.92 a - c 123.08 a - c 195.42 ab 93.91 a Ulocladium oudemansii ( BotryStop ) 234.50 a - c 154.00 a 244.42 cd 159.83 a 152.83 bc 113.17 a Streptomyces lydic us ( Actinovate ) 254.92 a - c 117.25 a - c 269.50 a - d 119.58 a - c 196.58 ab 89.25 ab Pseudomonas chlororaphis ( Zio ) 292.83 a 105.00 a - c 323.17 a 105.58 bc 219.92 a 82.25 ab 41 Untreated inoculated control 289.33 a 95.08 bc 318.50 ab 99.17 bc 210.00 a 76.42 ab x Disease assessment were done on 6, 13 and 20 - days post inoculation on 26 Sept, 3 and 10 Oct 2019 (Trial 1) and 24 and 31 Oct; 7 Nov 2019 (Trial 2). y Column cted LSD; P=0.05). 42 Figure 2 . w hen inoculated with Botrytis cinerea and treated with biorational products A: Untreated inoculated control, B: Streptomyces lydicus (Actinovate), C : Pseudomonas chlororaphis (Zio), D: Extract of S winglea glutinosa (Ecoswing) , E: Aureobasidium pullulans ( Botector) , F: F enhexamid (Decree) A F D C B E 43 Bi o rational evaluation on . In both trials, the disease assessment at the last observation and th e AUDPC data for the fungicide fenhexamid (Decree) showed effective control with an exception of foliar lesions in Trial 2. Data associated with the last disease assessment for both trials indicated that Bacillus amyloliquefa ciens ( Serifel ) , Pseudomonas ch lororaphis ( Zio ) , Aureobasidium pullulans ( Botector ) , and e xtract of Swinglea glutinosa ( Ecoswing ) were similar to the fungicide standard for the number of blighted leaves and lesions. The AUDPC data for these parameters indicated that A . pullulans (Trials 1 and 2), Bacillus subtilis ( Serenade Opti ) (Trial 2) and B . amyloliquefaciens (Trial 1) provided a level of efficacy similar to the fungicide fenhexamid . 44 Table 7 . Mean n umber of blighted leaves , foliar lesions and leaves with sporulating B otrytis ci nerea Pinto Premium Orange geranium treated with biorational products and a fungicide standard 20 days following inoculation . Treatment (Trade name/active ingredient) B lighted leaves (no.) Foliar lesions (no.) L eaves with sporulation Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Fenhexamid ( Decree ) 13.2 d 8.0 cd 14.6 d 10.2 bc 9.0 c 4.2 d x Bacillus amyloliquefaciens ( Serifel ) 16.0 cd 13.4 a - c 16.2 cd 15.0 ab 13.8 bc 11.8 a - c Pseudomonas chlororaphis ( Zio ) 16.6 cd 13.6 ab 19.2 b - d 15.2 ab 14. 2 bc 12.0 a - c Bacillus subtilis ( Serenade Opti ) 18.8 b - d 7.8 d 19.6 b - d 7.8 c 18.0 b 6.8 cd Aureobasidium pullulans ( Botector ) 19.2 b - d 13.4 a - c 21.8 b - d 14.4 ab 16.2 bc 10.4 a - c Extract . Swinglea glutinosa ( Ecoswing ) 20.0 b - d 12.8 a - d 21.0 b - d 13.0 a - c 16.8 bc 10.8 a - c Gliocladium catenulatum ( Prestop ) 20.8 b - d 16.4 a 23.0 bc 16.4 a 16.0 bc 14.6 a Bacillus mycoides ( LifeGard ) 22.4 bc 13.4 a - c 24.0 bc 13.4 a - c 21.2 ab 12.2 a - c Streptomyces lydicus ( Actinovate ) 22.4 bc 15.2 ab 24.2 ab 15.6 ab 19.2 b 14 .6 a Soybean and corn oil ( PureCrop1 ) 23.8 a - c 10.4 b - d 25.8 ab 11.4 a - c 19.6 b 8.8 b - d Ulocladium oudemansii ( BotryStop ) 25.4 ab 12.8 a - d 26.2 ab 12.8 a - c 21.4 ab 11.8 a - c Untreated inoculated control 31.2 a 14.2 ab 32.0 a 14.2 ab 29.0 a 13.6 ab x Colu 45 Table 8 . Area under disease progress curve (AUDPC) data for blighted leaves, foliar lesions and leaves with sporulating B . cinerea on Pinto Premium Orange geranium treated with bio rational products. Treatment (Trade name/active ingredient) AUDPC for blighted leav es AUDPC for foliar lesions AUDPC for l eaves with sporulati on x Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Fenhexamid ( Decree ) 95.2 d 67.2 b 113.4 d 75.6 cd 44.8 d 27.3 c y Bacillus amyloliquefaciens ( Serifel ) 129.5 cd 130.2 a 135.1 cd 139.3 ab 86.8 cd 112.0 ab Pseudomonas chlororaphis ( Zio ) 175.7 bc 161.7 a 236.6 ab 190.4 a 110.6 bc 134.4 a Bacillus subtilis ( Serenade Opti ) 169.4 bc 71.4 b 1 82.0 b - d 72.8 d 124.6 bc 57.4 b Aureobasidium pullulans ( Botector ) 158.9 b - d 113.4 ab 178.5 b - d 119.7 b - d 105.7 bc 88.9 ab Extract . Swinglea glutinosa ( Ecoswing ) 189.7 a - c 123.2 ab 214.2 ab 134.4 a - c 123.2 bc 92.4 ab Gliocladium catenulatum ( Prestop ) 17 9.9 bc 126.7 a 211.4 ab 136.5 ab 90.3 b - d 105.7 ab Bacillus mycoides ( LifeGard ) 177.8 bc 136.5 a 195.3 bc 142.8 ab 132.3 bc 110.6 ab Streptomyces lydicus ( Actinovate ) 204.4 ab 167.3 a 231.0 ab 181.3 a 137.2 ab 149.8 a Soybean and corn oil ( PureCrop1 ) 19 5.3 a - c 103.6 ab 221.9 ab 112.7 b - d 126.7 bc 84.0 ab Ulocladium oudemansii ( BotryStop ) 203.0 ab 139.3 a 231.7 ab 160.3 ab 114.8 bc 113.4 a 46 Untreated inoculated control 247.1 a 133.7 a 280.0 a 149.8 ab 184.8 a 107.1 ab x Disease as sessment were done on 6, 13 and 20 - days post inoculation on 26 Sept, 3 and 10 Oct 2019 (Trial 1) and 24 and 31 Oct; 7 Nov 2019 (Trial 2). y Means with the same letter are not significantly different ( =0.05) based on F - test. 47 Figure 3 . Botrytis cinerea and treated with biorational products. A: Untreated control, B: Pseudomonas chlororaphis (Zio), C: Bacillus subtilis (Serenade Opti), D: Aureobasidium pullulans ( Botector), E: Gliocladium catenulatum ( Prestop), F: F enhexamid (Decree) A F D C B E 48 DISCUSSION Botrytis blight requires intensive management efforts to reduce crop loss of floriculture crops (Grinstein et al., 1997) . While all geranium cultivars included in our trial were susceptible to B. cinerea , significant differences were observed. Disease incidence . In a previous study, an i vy geranium accession 86 - 23 - 1 (diploid, P. peltatum P. x hortorum ) had consistently high levels of res istance co mpared to P. x hortorum ) (Uchneat, et al., 1999b) . In our study, we found that the i vy geranium ( P. peltatum ) was less susceptible to B. cinerea than zonal geranium s ( P. x hortorum ) cultivar s . Among t he P. x hortorum cultivars included in this trial, there were significant differences in resistance to B. cinerea . Differences in B. cinerea susceptibility among cultivar s has been observed for geranium ( Uchneat , et al., 1999a, 1999b), petunia ( Krahl and Randle, 1999), lisianthus (Wegulo and Vilchez, 2007), and cut roses (Hammer and Evensen, 1994; Muñoz et al., 2019) . Host resistanc e to B . cinerea has been attributed to genetics, rate of senescence, structural defense, secondary metabolites and d efenses accelerated by hormone production (E lad and Evensen, 1995) . Senescent plant tissues is readily invaded by the pathogen so changes related to senescence can play a role in host resistance (Elad and Evensen, 1995) . Defense may be media ted through jasmonic acid, salicylic acid, abscisic acid and ethylene signaling pathway s which are li nked in a complex network (AbuQamar et al., 2 017) . Nitric oxide has an important role in resistance of geranium to B . cinerea with early nitric oxides bursts and production of secondary nitric oxide stimulating noncell - death - associated defense (Floryszak - wieczorek et. al, 2007) . 49 I nconsistencies among t he geranium cultivars were observed in this research when t he experiment w as repeated using the similar procedures. I nconsistencies among cultivars in their susceptibility to B. cinerea was noted in studies including petunia (Krahl and Randle 1999 ) and lisianthu s ( Wegulo and Vilchez, 2007) f ference in disease pressure between the two trials may be due to variation in the environment. During the incubation period there were differences in temperature between the trials . In Trial 1, a verage temperature was 18.4 0 C with minimum/maximum temperatur es of 12.2 / 21.9 0 C. In Trial 2, the average temperature was 25 0 C with minimum/maximum temperatures of 21.3 / 32.7 0 C. RH was greater than 90% in both trials. The optimum temperature for B. cinerea conidial germination is 22 to 25 0 C with RH > 90% (Salina s et al. , 1989) with infection occur ring between 15 to 25 0 C (Jarvis, 1989) . Conidial g ermination decreases at temperature > 25 0 C and is inhibited at 30 0 C (Salina s et al. , 1989) . Th us, the reduced disease pressure i n Trial 2 may have been the result of the temperature exceeding the optimum requirements for disease development. Although the flowers remained on the plant during our assessment, we did not evaluate flower blighting as the timing of flowering was inconsistent among the cultivars. While Uchneat et al. (1999a) determined that there is no correlation between floral and foliar re sistance . Flower petals are highly susceptible to infection and may serve as the initial source of inoculum for foliar infection ( (Williamson et al., 2007) . Several biorational products evaluate d in our study effectively limited Botrytis blight in geranium. In Trial 1, Aureobasidium pullulans (Botector) , Bacillus amyloliquefaciens (Serifel) and Gliocladium catenulatum (Prestop) were effective when tested on a highly susceptible and 50 moderately res istant geranium cultivar according to the AUDPC values for blighted leaves, foliar lesions and leaves with sporulating B. cinerea . G. catenulatum has antagonistic ac tivity through antibiosis and mycoparasitism of B. cinerea conidia and germ tube which limi t s the disease whereas A. pullulans competes with the pathogen for nutr ients (Castoria et al., 2001; Jacometti et al., 2010; Pal and Gardener, 2006; Vidhyasekaran , 2004 ) . Elmhirst et al. (2011 ) also reported that on greenhouse geraniums , G. catenulatum effectively reduced d isease incidence and severity. This product also effectively limite d B. cinerea on greenhouse tomatoes and cucumber through an antagonistic mode of action (Dik et al., 1999; Utkhede and Mathur, 2006) . Applications of Gliocladium roseum also decreased disease i ncidence in greenhouse cyclamen (Köhl et al. , 1998) . In th e present study , A. pullulans effectively limited B . cinerea in both geranium cultivars. In a previous stud y, A. pullulans was moderate ly to highly effective for B . cinerea control on greenhous e tomato and cucumber and reduced the number of diseased fruits and stem lesions ( Dik and Ela d , 1999) . Efficacy of A. pullulans for B. cinerea control has also been reported on strawberry (Lima et al. , 1997 ; Sylla et al., 2015) and grape crops (Fedele et al., 2020; Pertot et a l., 2017) . Pseudomonas chlororaphis (Zio) and t he Bacillus p roducts including B. subtilis (Serenade Opti) and B. mycoides (LifeGard) effectively controlled Botrytis blight on the moderately resistant date for t he number of blighted leaves, foliar lesions and number of le aves with pathogen sporulati on . Bacillus species including B. subtilis, B. amyloliquefaciens and B. mycoides limit B. cinerea through induction of systemic acquired resistance (Choudhary and Johri, 2009 ) , mycoparasitism , and antibiosis (Pal and Gardener, 2006; Paulitz and Belanger, 2001) . According to AUDPC data for leaves with 51 sporulating B. cinerea , all tested products effectively limited disease except Streptomyces lydicus (Actinov ate) for in Trial 1. Biorational products did not provide c onsistent results between trials. This has been reported by others as the suppression of B. cinerea is highly affected by the environment which influences the surv ival of the biocontrol agents on the phyllosphere and their ability to control the pathogen (Guetsky et al., 2001; Shtienberg and Elad, 1997) . Combining two or more biocontrol products with different mechanism may reduce the variability of the biocontrol products and effectively control Botrytis blight (Guetsky et al. , 2001; Guetsky et al., 2002; Pertot et al., 2017) . In summary, results from the present study indicate that moderately resistant to B. cinerea were highly susceptible. None of the cultivars included in this study were immune to B. cinerea as all became infected. Cultivars with a moderate resistance could be combine d with biorational products including Aureobasidium pullulans (Botector), Gliocladium c atenulatum (Prestop) and Bacillus amyloliquefaciens (Serifel) to achieve a sustainable disease management strategy . Cultivars that are hig hly susceptib le to B. cinerea r esistance may need to be protected using conventional fungicides along with other contr ol strategies . 52 LITERATURE CITED 53 LITERATURE CITED 1. AbuQamar, S., Moustafa, K., and Tran, L. S. P. 2017. Mechanisms and strategies of plant defense against Botrytis cinerea . Crit. Rev. Biotechnol . 37 : 262 274. 2. Castoria , R., De Curtis, F., Lima, G., Caputo, L., Pacifico, S., and De Cicco, V. 2001. Aureobasidium pullulans (LS - 30) an antagonist of postharvest pathogens of fruits: Study on its modes of ac tion. Postharvest Biol . Technol . 22:7 17. 3. Chandel, S., and Kumar, V. 2018. D iseases of geranium and their management. 223 - 242 . 4. Choudhary, D. K., and Johri, B. N. 2009. Interactions of Bacillus spp . and plants - With special reference to induced systemic resistance (ISR). Microb iol. Res . 164:493 513. 5. Dean , R ., Van Kan , J . A . L., Pretorius , Z . A ., Hammond - Kosack , K . E ., Di Pietro , A ., Spanu , P . D ., Rudd , J . J ., Dickman , M ., Kahmann , R ., Ellis , J ., and Foster , G . D. 2012. The Top 10 fungal pathogens in mole cular plant pathology. Mol . Plant Pathol. 134 : 414 430. 6. Dik, A. J., and Elad, Y. 1999. Comparison of antagonists of Botrytis cinerea in greenhouse - grown cucumber and tomato under different climatic conditions. E ur . J . Plant Pathol . 105 : 123 137. 7. Dik, A. J ., Koning, G., and Köhl, J. 1999. Evaluation of microbial antagonists for biological control of Botrytis cinerea stem infection in cucumber and tomato. Eur . J . Plant Pathol . 105 : 115 122. 8. Elad , Y., and Evensen, K. 1995. Physiological a spects of r esistance to Botrytis cinerea . Phytopatholog y. 85 : 637 343 . 9. Elad, Y., and Shtienberg, D. 1995. Botrytis cinerea in greenhouse vegetables: chemical, cultural, physiological, and biological controls and t heir integration. Integr . Pest Manag . Rev . 1:15 29. 10. Elmhirst, J. F., Haselhan, C., and Punja, Z. K. 2011. Evaluation of biological control agents for control of botrytis blight of geranium and powdery mildew of rose. Can . J . Plant Pathol . 334:499 505. 11. Fedele, G., González - Domínguez, E., Ammour, M. S., Languasco, L., and Rossi, V. 2020. Reduction of Botrytis cinerea colonization of and sporulation on bunch trash. Plant Dis . 104 : 808 816. 54 12. Floryszak - wieczorek, J., Arasimowicz, M., Milczarek, G., Jelen, H., and Jackowiak, H. 2007. Only an early nitric oxide burst and the following wave of secondary nitric oxide generation enhanced effective defence responses of Pelargonium to a necrotrophic pathogen . New Phytol. 175: 718 730. 13. Gerlagh, M., Amsi ng, J. J., Molhoek, W. M. L., Bosker - Van Zessen, A. I., Lombaers - Van Der Plas, C . H., and Köhl, J. 2001. The effect of treatment with Ulocladium atrum on Botrytis cinerea - attack of geranium ( Pelargonium zonale ) stock plants and cuttings. Eur . J . Plant Pathol . 107:377 386. 14. Grinstein, A., Riven, Y., and Elad, Y. 1997. Improved chemical control of botrytis blight in roses. Phytoparasitica . 25:87 92. 15. Guetsky, R., Shtienberg, D., Elad, Y., and Dinoor, A. 2001. Combining biocontrol agents to reduce the variability of biological control. Phytopathology . 91:621 627. 16. Guetsky, Ruth, Shtienber g, D., Elad, Y., Fischer, E., and Dinoor, A. 2002. Improving biological control by combining biocontrol agents each with several mechanisms of disease suppre ssion. Phytopathology . 92:976 985. 17. Hahn, M. 2014. The rising threat of fungicide resistance in pla nt pathogenic fungi: Botrytis as a case study. J . Chem . Bio l. 7 : 133 141. 18. Hammer, P. E., and Evensen, K. B. 1994. Differences between rose cultivars in susceptibility to infection by Botrytis cinerea . Phytopathology. 84:1305 - 1312. 19. Hausbeck, M. K. , and Harl an, B. 2020. Recommendations for Botrytis f ungicides for 2020. MSU Extension , Floriculture and Greenhouse Crop Production . 20. Hausbeck, M. K., and Moorman , G. W. 1996. Managing Botrytis in g reenhouse - g rown f lower Crops. Plant Dis. 80:1212 - 1219. 21. Hausbeck, M. K., and Pennypacker, S. P. 1991. influence of g rower activity on concentrations of airborne conidia of Botrytis cinerea among geranium cuttings. Plant Dis . 75 : 1236 1243. 22. Horst, L. E., Locke, J., Krause, C. R., McMahon, R. W., Madden, L. V., and Hoitink, H. A. J. 2005. Suppression of Botrytis blight of begonia by Trichoderma hamatum 382 in peat and compost - amended potting mixes. Plant Dis . 89:1195 1200. 23. Jacometti, M. A., Wratten, S. D., and Walter, M. 2010. Review: Alternatives to synthetic fungicides for Botrytis cinerea management in vineyards. Aust . J . Grape Wine R . 16:154 172. 55 24. Jarvis, W. R. 1989. Managing Diseases in Greenhouse Crops. Plant Dis. 73:190 - 194. 25. Jiang, C., Liao, M., Wang, H., Zheng, M., and Xu, J. 2018. Bacillus velezensis , a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea . Biol . Control . 126:147 157. 26. Kim, J. - O., Shin, J. - H., Gumilang, A., Chung, K., Choi, K. Y., and Kim, K. S. 2016. E ffectiveness of different classes of f ungicides on Botr ytis cinerea causing gray mold on fruit and vegetables. Plant Pathol . J . 32 : 570 574. 27. Köhl, J., Gerlagh, M., De Haas, B. H., and Krijger, M. C. 1998. Biological control of Botrytis cinerea in cyclamen with Ulocladium atrum and Gliocladium roseum under comm ercial growing conditions. Phytopathology . 88 : 568 575. 28. Krahl, K. H., and Randle, W. M. 1999. Resistance of petunia phenotypes to Botrytis cinerea . HortScience . 34:690 692. 29. Lima, G., Ippolito, A., Nigro, F., and Salerno, M. 1997. Effectiveness of Aureobas idium pullulans and Candida oleophila against postharvest strawberry rots. Postharvest Biol . Technol . 10 : 169 178. 30. Moorman, G. W., and Lease, R. J. 1992. B enzimidazole - and d icarboximide - resistant Botrytis cinerea from P ennsylvania greenhouses. Plant Dis . 76 : 477 480. 31. Muñoz, M., Faust, J. E., and Schnabel, G. 2019. Characterization of Botrytis cinerea from commercial cut flower roses. Plant Dis . 103 : 1577 1583 . 32. Olson, H. A., and Benson, D. M. 2007. Induced systemic resistance and the role of binucleate Rhi zoctonia and Trichoderma hamatum 382 in biocontrol of Botrytis blight in geranium. Biol . Control. 42(2):233 - 241. 33. Pal, K. K., and Gardener, B. M. 2006. Biological control of plant pathogens. The Plant Health Instructor. 2 : 1117 - 1142 . 34. Paulitz, T. C., and Be langer, R. R. 2001. Biological c ontrol in g reenhouse s ystem. Annu . Rev . Phytopathol . 39:103 133. 35. Pertot, I., Giovannini, O., Benanchi, M., Caf fi , T., Rossi, V., and Mugnai, L. 2017. Combining biocontrol agents with different mechanisms of action in a stra tegy to control Botrytis cinerea on grapevine . Crop Prot. 97:85 93. 36. Punja, Z. K., an d Utkhede, R. S. 2003. Using fungi and yeasts to manage vegetable crop diseases. Trends Biotechnol . 21:400 407. 56 37. Salinas, J., Glandorf, D. C. M., Picavet, F. D., and Verhoe ff, K. 1989. Effects of temperature, relative humidity and age of conidia on the inci dence of spotting on gerbera flowers caused by Botrytis cinerea . Neth . J . Plant Pathol . 95 : 51 64. 38. Samarakoon, U. C. , Schnabel, G., F aust, J. E., Bennett, K., Jent, J., Hu , M. J., Basnagala, S., and Williamson , M. 2017. First report of resistance to multiple chemical classes of fungicides in B otrytis cinerea , the causal agent of gray mold from greenhouse - grown petunia in F lorida . Plant Dis. 101:1052. 39. Shtienberg, D., and El ad, Y. 1997. Incorporation of weather forecasting in integrated, biological - chemical management of Botrytis cinerea . Phytopathology . 87:332 340. 40. Simko, I., and Piepho, H - P. 2012. The area under the disease progress stairs: Calculation, advantage, and appl ication. Phytopathology 102:381 - 389. 41. Sylla , J., Alsanius, B. W., Krüger, E., and Wohanka, W. 2015. Control of Botrytis cinerea in strawberries by biological control agents applied as single or combined treatments. Eur . J . Plant Pathol . 143 : 461 471. 42. Uchne at, M. S., Spicer, K., and Craig, R. 1999 a . Differential response to floral infection by Botrytis cinerea within the genus Pelargonium . HortScience. 34:718 - 720. 43. Uchneat, M. S., Zhigilei, A., and Craig, R. 1999 b . Differential Response to Foliar Infection w ith Botrytis cinerea within the Genus Pelargonium . J . Am . Soc . Hortic . Sci . 124:76 80. 44. USDA - National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. Retrieved from: USDA - National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. Retrieved from: ht tps://www.nass.usda.gov/Publications/Todays_Reports/reports/floran19.pdf 45. Utkhede, R. S., and Mathur, S. 2006. Preventive and curative biological treatments for control of Botrytis cinerea stem canker of greenhouse tom atoes. Bio l . Control . 51:363 373. 46. Vid hyasekaran, P. 2004. Biological control Microbial pesticides. In: Concise encyclopedia of plant pathology. Food Products Press. Binghamton . USA:239 270. 47. Wegulo, S. N., and Vilchez, M. 2007. Evaluation of Lisianthus Cultivars for Resistance to Botrytis c inerea . Plant Dis . 91:997 1001. 48. Williamson, B., Tudzynski, B., Tudzynski, P., and Van Kan, J. A. L. 2007. Botrytis cinerea : The cause of grey mould disease. Mol . Plant Patho l . 8:561 580. 57 49. Yourman, L. F., and Jeffers, S . N. 1999. Resistance to Benzimidazole and Dicarboximide Fungicides in Greenhouse Isolates of Botrytis cinerea . Plant Dis . 83:569 575. 58 CHAPTER 2. MANAGEMENT OF BOTRYTIS CINEREA IN PETUNIA USING CULTIVAR RESISTANCE AND BIORATIONAL PRODUCTS 59 ABSTRACT Botrytis cinerea , causes blight on the leaves, stems, and flowers of p etunia ( Petunia x hybrida ), a popular annual bedding plant. Our objectives w ere to evaluate: (i) petunia cultivars for suscepti bility to Botrytis blight and (ii) biorationals t hat limit Botrytis blight. Thirteen traditional and spreading type (wave) petunia cultivars were selected . Ten biorational products were evaluated for control of Botrytis blight and compared to the standard fungicide fenhexamid and an untreated control. The area under the disease progress curve (AUDPC) was calculated. had significantly higher disease severity and AUDPC values than in the trial s . According to AUDPC d susceptible to B. cinerea and had a disease severity rating and Aureobasidium pullulans ( Botector ) and G liocladium catenulatum (Prestop) provided B. cine rea control similar to fungicide standard fenhexamid (Decree) in both trials. Applications of Pseudomonas chlororaphis (Zio) resulted disease severity ratings and AUDPC values similar to the fungicide standard for both trials with the exception of AUDPC data in Trial 1. According to final disease severity assessment, treatment with soybean and corn oil (PureC rop1) and Ulocladium oudeman sii ( BotryStop ) and B acillus mycoides ( LifeGard ) also provided control similar to the fungicide standard but was not significantly different from untreated control. Results from this study illustrate that certain biorational pro ducts can limit B. cinerea w hen used in conjunction with a cultivar that has disease resistance . 60 INTRODUCTION Petunia ( Petunia x hybrida ) is one of the most po pular annual bedding plants and is availble in a range of flower color s and growth habits. In 2018, the total U.S. sales of petunia sold in pots, flats or hanging baskets was $141.7 million (USDA - NAS, 2019). petunia has become popular due to its vigorous nature and trailing growth which are ideal for hanging baskets. Botrytis blight i s one of the most important disease of greenhouse ornamentals and is incited by the airborne necrotrophic fungus Botrytis cinerea (teleomorph: Botryotinia fuckeliana ) . Considered the second most destructive pathogen in the world (Williamson et al., 2007 ; Dean et al. , 2012) , B. cinerea affects more than 200 crop species causing blossom and leaf blight, stem canker, damping off, bud, crown and fruit rot ( Hausbeck and Moorman, 1996 ; Moyano et al., 2004; Williamson et al., 2007 ; Hahn, 2014 ; Jiang et al., 2018 ) . B. cinerea produc es grey masses of conidia on the surface of infected plant tissue which i s diagnostic ( Punja and Utkhede, 2003 ; Wil liamson et al., 2007 ). Production of ornamentals in the greenhouses favor s grey mold as warm temperatures, high relative humidity, free moisture, and a lack of air exchange provide favorable environmental conditions for the pathogen (Elad and Shtienberg, 1995; Paulitz and Belanger , 2001) . B . cinera m ay ente r the greenhouse through young seedlings and cutting s which later forms a source of ino culum in the production greenhouse (Dik and Wubben, 200 4 ) . Dispersal o f conidia in the greenhouse occurs through air current or water splash ( Jarvis, 1989 ) and the peak atmospherical conidial concentration was often associated with grower activity including watering, fertilization, pesticide application and harvesting cuttin gs (Hausbeck and Pennypacker, 1991) . 61 F lo wer infection i s the major concern of producers as the lesions render them unsuitable for marke ting. Latent infections may occur during production and become active during storage or transportation (Dik and Wubben, 2004) . For instance, asymptomatic petunia plants may har bor latent infections which develop during the cool moist conditions during ship ping. W hen retailers receive the plants , they are severely diseased with wilted and necrotic flowers (Samarakoon et al. , 2016 ) . Resistance to B . cinerea was observed by Kra hl and Randle ( 1999) on select petunia phenotypes but resistant cultivars are not commercially available. Cultural control of Botrytis blight includes sanitation, heating and venting, minimiz ing the d uration of leaf wetness, increased plant spacing and ai r circulation (Jarvis, 1989 ; Hausbeck and Moorman, 1996 ; Elad, 2016 ) . R emo ving dead and decaying plant parts exclude s the source of the inoculum and delay s the onset of the disease (Elad and Shtienberg, 1995) . H eating and vent ing the greenhouse reduce s the relative humidi ty and duration of dew periods (Elad, 2016; Elad and Shtienberg, 1995; Hausbeck et al. , 1996) . Application of biorational s or fungicides is often needed as an additional control measure for Botrytis blight . Multisite and site - speci fic fungicides (Hahn, 2014) have been relied upon to limit the disease in the greenhouse but fungicide resistance to single or multiple chemical classes has been noted in several cropping systems including strawberry (Fernández - Ortuño et al., 2015; Hu et al., 2016) , grape (Bertetti et al., 2 020; Saito et al., 2019) , greenhouse cucumber and tomato (Moyano et al., 2004) , cut rose s (Muñoz et al., 2019) , and petunia (Samarakoon et al., 2017). B. cinerea from g reenhouse grown cut roses w as r esistant to four different classes of fungicides (Muñoz et al., 2019) . Samarakoon et al. (2017) found that the pathogen from diseased petunias w as resistant to s ix different fungicides classes. Use of biorationals could decrea se reliance on 62 fungicide s and delay B. cinerea resistance . Yeast s ( Pichia spp ., Candida spp. ) (Jacometti et al., 2010 ) , b acteria ( Bacillus spp., Pseudomonas spp.) and filamentous fungi ( Ulocladium spp. , Gliocladium spp., Trichoderma spp.) ha ve effectively control led B. cinerea on different crops (Jacometti et al., 2010; Paulitz and Belanger, 2001) . Specifically, biorationals have proven effective in greehouse tomato, cucumber ( Dik et al., 19 99 ), pepper (Jiang et al., 2018) and ornamentals including be gonia (Horst et al., 2005) and geranium (Olson and Benson, 2007 ) . B iorationals offer various modes of action including competition for space and nutrients , parasitism, antibiosis, and induction of systemic acquired resistance (Choudhary and Johri, 2009; Pal and Gardener, 2006; Pa ulitz and Belanger, 2001) . Our objective was to evaluate selected petuni a cultivars for susceptibility to Botrytis blight and the ability of biorational products to limit disease. Integrating host resistance with effective biorational products could offer growers sustainable control options . MATERIAL AND METHODS C ultivar scre ening: The following p etunia s ( Petunia x hybrida ) included the following: , ( Ball Horticultural Company, IL, USA ) . Seeds were sown in 128 - cell plug trays containi ng soilless root medium ( Suremix Perlite, Michigan Growers Products Inc, Galesburg, MI ) on 20 Dec 2018 and incubated in the Plant Science G reenhouse at M ichigan State University (MSU) , East Lansing, MI. S eedlings were transplanted 42 days after seeding (9 Oct 2019) into square pots (10*10 cm 2 ) filled with soilless root medium and fertilized daily with 200 ppm water - soluble 20:20:20 NPK fertilizer ( ICL Spec ialty fertilizers, Dublin, OH ). 63 A B . cinerea isolate from geranium was cultured on potato dextrose ag ar (PDA) media and grown under florescent light under laboratory conditions to induce sporulation . I ron baskets were sanitized (10% solution, Clor o x germ icidal bleach, The Clor o x company, Oakland, CA) and place d in side translucent plastic bags (21 cm x 5.5 cm x 38 cm) containing water at the bottom to achieve high relative humidity ( RH ) . Eight plants , single - plant replication per treatment, from each culti var were selected and placed inside the basket and arranged in completely randomized design on th e bench in 80% shaded greenhouse at MSU . T he conidial suspension was prepared by dislodging 11 - day - old B. cinerea culture s flooded with distilled water and str ained through cheesecloth. The conidial c oncentration was standardized to 1x10 6 conidia/ml soluti on with a hemocytometer. P lants were inoculated by spraying the B. cinerea conidial suspension on 12 Mar 2019 o n the plant surface uniformly with a hand spraye r until run off. Inoculated plants were incubated by closing the translucent p lastic bags with a rubber band to provide high RH . A W atchdog data logger (Spectrum technologies Inc., Aurora, IL) w as installed in one basket to monitor daily temperature and R H inside the bag. The experiment was conducted for 21 days (12 Mar to 2 Apr 2019) with disease as sessed three times at 7 - day intervals ( 19, 26 Mar and 2 Apr) . A verage temperature of 20.7 0 C was recorded during the incubation with max./min. temperature of 22 .5 0 C/20.3 0 C. The experiment was repeated twice (9 to 30 Apr and 13 Sept to 4 Oct 2019) using the procedure as previously described with four additional cultivars including for a to tal of 13 cul tivars . Plants were inoculated with conidial suspension of Botrytis on 9 Apr and 12 Sept for Trial 2 and 3, respectively. Disease assessment w as done 7, 14 and 21 - days post inoculation on 16, 23 and 30 Apr (Trial 2) and 20, 27 Sept and 4 Oct 2 019 (Trial 3) . Average temperature during the incubation period were 22.8 0 C and 23.7 0 C with 64 max./min. temperature of 33.4 0 C/17.2 0 C and 30.8 0 C/22.1 0 C for Trial 2 and 3 respectively. Efficacy of bio rational products : , identified as one of the less susceptible petunia cultivars to B. cinerea in our studies was selected. S eed was sown i n the 128 - cell plug trays in the Plant Science G reenhouse at MSU , East Lansing, MI on 28 Aug 2019 and seedlings were transplanted six week s later (9 Oct 2019) into square pots (10*10 cm 2 ) filled with soilless root medium (Suremix Perlite, Michigan Growers Products Inc, Galesburg, MI ). The transplanted plants were fertilized daily with 200 ppm water - soluble 20 - 20 - 20 NPK fertilizer ( ICL Specialty fertil izers, Dublin, OH ). Ten bio rational products and the standard f ungicide D ecree (fenhexamid) were each applied at 7 - day intervals using a hand compressed air sprayer (Table 1). Three application (14, 21 and 28 Nov) were made for each product with the except ion of Gliocladium catenulatum ( PreStop) which was applied one time as the label specifies a 21 - day application interval. F ive replications each comprising of a single plant , for a total of 60 plants were arranged in a completely randomized design on a sha ded (80%) bench at the Plant Science G reenho uses at MSU, East Lansing, MI . The experiment was conducted from 14 Nov to 5 Dec 2019. Conidial suspension of B. cinerea conidia (10 6 co nidia/ml) was applied one day following treatment (15 Nov) by spraying the c onidial suspension to the plants until runoff using a hand sprayer. Disease was asses sed 6 , 1 3 and 2 0 - days post inoculation on 21, 28 Nov and 5 Dec . A watchdog data logger was used to monitor the environmental conditions as described previously. M ax./min. temperature inside plastic bag were 24.1 0 C /15.8 0 C with average of 19.5 0 C during the period of experiment. The experiment was repeated from 14 Jan to 4 Feb 2020 using the same procedure as described above. Plants were inoculated with B. cinerea (15 Jan) an d treatments were applied for three times (14, 21 and 28 Jan). Disease was assessed at 7 - day interval on 21, 28 Jan and 4 Feb 2020 . 65 Table 9 . Biorational produc ts and a standard fungicide evaluated for efficacy against Botrytis cinerea on petunia. Product Active Ingredient Registrant Rate/ 100 gal Actinovate® SP Streptomyces lydicus WYEC108 (0.037%) Novozymes BioAg Inc. 12 oz Botector® Aureobasidium pullulans strain DSM 14940 (40%), DSM 14941 (40%) Bio - ferm 10 oz Ulocladium oudemansii strain U 3 BioWorks , Inc. 4 lb Extract of Swinglea glutinosa (82%) Gowan Company 2 pt Bacillus mycoides (40%) Certis USA 4.5 oz Prestop® WP Gliocladium catenulatum strain J1446 (32%) Danstar Ferment AG 70 oz PureCrop1 Soybean oil (10%), C orn oil (5%) PureCrop1 200 oz Serenade Opti® WP Bacillus subtilis QST713 (26.2%) Bayer CropScience Inc. 20 oz Serifel® Bacillus amyloliquefaciens strain MBI600 (11%) BASF Corporation 16 oz Pseudomonas chlororaphis strain AFS009 SePRO Corporation 1 00 oz Decree® 50 WDG Fenhexamid (50%) SePro Corporation 1 lb 66 Disease assessment : T he total diseased area (%) o f each plant assessed visually using a scale of 0 to 10 (0 = no disease , 1 = 1 - 10%, 2 = 11 - 20%, 3 = 21 - 30%, 4 = 31 - 40%, 5 = 41 - 50%, 6 = 51 - 60%, 7 = 61 - 70%, 8 = 71 - 80%, 9 = 81 - 90% and defoliation , 10 = >91% and plant death (Elmhirst et al., 2011) . A ssessment s were conducted 7, 14 and 21 - day s post inoculation. The area under disease progression curve (AUDPC) was calculated to express the cumulative disease severity using the formula AUDPC = i + y i+1 )/2] x (t i+1 t i ) where y i is the assessment of disease at ith observation, t i is the time (days) at the i th observation and n is the total number of observations (Simko and Piepho, 2012) . Statistical Analysi s : Data were analyzed with a one - way ANOVA using PROC GLIMMIX procedure on SAS Statistical Analyzing Software ( SAS Institute Inc., Cary, NC, 2013 ) for disease severity and determined least square means among the treatments . Normal distribution of the data was met when checked through residual plots and h omogeneity analysis rea under disease progress curve (AUDPC) for disease severity w as calculated from the three ratings . Statistical di fferences a mong treatments in all trials w ere d et ermined east S ignificant D ifference t - test (P=0.05). 67 RESULTS Cult i var screening : According to the final disease assessment and AUDPC data, had significantly less disease than in each t rial. vels and AUDPC data in , had significantly more disease according to the final disease assessment. According to AUDPC k B. cinerea and had a 68 Table 10 . Dise ase severity on petunia cultivars in the greenhouse observed 21 days following inoculation with Botrytis cinerea . Cultivar s Disease Severity z Trial 1 Trial 2 Trial 3 Tidal Wave Cherry 6.88 a 4.75 bc 6.67 a y Success Burgundy - x 8.00 a 6.67 a Debonair Lime Green 4.63 bc 7.00 a 6.33 ab Wave lavender - 5.25 b 5.67 a - c Easy Wave Red Improved 4.00 cd 4.75 bc 5.17 bc Sophistica Blackberry 4.13 cd 3.00 d 5.00 c Wave Purple Classic 3.25 d 5.50 b 4.83 c Shock Wave Coconut 7.38 a 5.25 b 4.67 cd Tidal Wave Silver 4.88 bc 4.38 bc 4.67 cd Easy Mix Flag - 3.63 cd 4.50 c - e Ramblin Red - 5.25 b 4.50 c - e Shock Wave Red 4.63 bc 4.88 b 3.50 de Easy Wave Blue 5.25 b 4.88 b 3.33 e z Disease rating scale 0 to 10 (0= no disease, 1= 1 - 10% , 2= 11 - 20% , 3 =21 - 3 0% , 4 =31 - 40% , 5= 41 - 50% , 6= 51 - 60% , 7= 61 - 70% , 8= 71 - 80% , 9= 81 - 90% and defoliation, 10 = >91% blighting and plant death . y P=0.05). x Petunia cultivars not included in T rial 1. 69 Table 11 . Area under disease progress curve (AUDPC) for disease severity on petunia cultivars in the greenhouse when inoculated with Botrytis cinerea . Cultivars AUDPC for disease s everity z Trial 1 Trial 2 Trial 3 Tidal Wave Cherry 76.56 a 56 .44 c - e 71.75 a y Debonair Lime Green 49.00 cd 67.37 bc 70.00 a Success Burgundy - x 81.81 a 57.17 b Wave Lavender - 66.06 b - d 51.33 bc Shock Wave Coconut 80.94 a 60.81 b - e 50.75 bc Easy Wave Red Improved 40.25 d 50.75 ef 50.17 bc Tidal Wave Silver 55. 13 bc 54.69 de 47.83 bc Sophistica Blackberry 44.63 cd 33.25 g 45.50 cd Easy Mix Flag - 37.19 g 43.75 cd Wave Purple Classic 37.63 d 71.31 ab 42.58 cd Ramblin Red - 58.19 c - e 40.83 c - e Shock Wave Red 49.00 cd 41.12 fg 35.0 de Easy Wave Blue 63.88 b 5 8.62 c - e 31.50 e z Disease assessment were done on 7, 14 and 21 - days post inoculation on 19, 26 Mar and 2 Apr (Trial 1), 16, 23 and 30 Apr (Trial 2) and 20, 27 Sept and 4 Oct 2019 (Trial 3). y Column means with a letter in common are not statistically diffe P=0.05). x Petunia cultivars not included in T rial 1. 70 Figure 4 . Highly susceptible (A, B) and least susceptible (C, D) petunia cultivars observed 2 1 days following the inoculation with Botrytis cinerea . A: Tidal Wave Cherry , B: Success Burgundy , C: Shock Wave Red , D: Sophistica Blackberry A B C D 71 Efficacy of bio rational products : T he f inal disease severity rating for the untreated control was 4.0 to 5.4 for Trials 1 and 2, respectively . T he fungicide standa rd Decree (fenhexamid) resulted in significantly less disease than the untreated control according to disease severity in Trial 1 and AUDPC data in Trial 2. According to disease severity ratings and AUDPC data, applications of Aureobasidium pullulans ( Bote ctor ) and Gliocladium catenulatum ( Prestop ) limited disease and was similar to the fungicide standard fenhexamid (Decree) for both trials. Treatments of Pseudomonas chlororaphis ( Zio ) provided disease severity ratings and AUDPC data similar to the fungicid e standar d fenhexamid for both trials with the exception of AUDPC data in Trial 1. Many of the biorational products provided control similar to the fungicide standard fenhexamid in both trials according to the final disease severity. According to disease s everity a ssessments, s oybean and corn oil ( PureCrop1 ) , Ulocladium oudemansii ( BotryStop ) , and Bacillus mycoides ( LifeGard ) provided control similar to the fungicide Decree standard in both trials; these products were also similar to the untreated control. 72 Table 12 . D isease severity Shock W ave Red petunia when inoculat ed with B otrytis cinerea and treated with biorational products and a fungicide standard. Treatment s Disease severity z AUDPC y Tria l 1 Trial 2 Trial 1 Trial 2 Fenhexamid ( Decree ) 2.8 cd 4.4 a - c 24.5 de 41.3 d x Aureobasidium pullulans ( Botector ) 3.2 b - d 3.8 c 32.2 cd 43.4 cd Pseudomonas chlororaphis ( Zio ) 3.2 b - d 4.0 bc 35.0 a - c 46.2 b - d Gliocladium catenulatum ( Prestop ) 2.2 d 4.4 a - c 21.0 e 46.9 b - d Bacillus mycoides ( LifeGard ) 3.8 a - c 4.6 a - c 35.0 a - c 47.6 b - d Ulocladium oudemansii ( BotryStop ) 3.2 b - d 4.8 a - c 35.0 a - c 52.5 a - c Bacillus amyloliquefaciens ( Serifel ) 4.8 a 5.4 a 44.1 a 52.5 a - c Soybean and corn oil ( PureCrop1) 3.8 a - c 4.8 a - c 34.3 bc 53.9 ab Extract of Swinglea glutinosa ( Ecoswing ) 4.4 a 4.8 a - c 34.3 bc 54.6 ab Bacillus subtilis ( Serenade Opti ) 4.6 a 5.0 a 42.0 ab 56.0 ab Streptomyces lydicus (Actinovate) 4.2 ab 5.4 a 41.3 a - c 58.1 a Untreated inoculated contr ol 4.0 ab 5.4 a 32.9 b - d 58.8 a z Disease rating scale 0 to 10 (0 = no blighting , 1 = 1 - 10% blighting , 2 = 11 - 20% blighting , 3 = 21 - 30% blighting , 4 = 31 - 40% blighting , 5 = 41 - 50% blighting , 6 = 51 - 60% blighting , 7 = 61 - 70% blighting , 8 = 71 - 80% blighting , 9 = 81 - 90% blighting and defoliation , 10 = >91% blighting and plant death . y Disease was assessed 6, 13 and 20 - days post inoculation on 21, 28 Nov and 5 Dec 2019 (Trial 1) and 21, 28 Jan and 4 Feb 2020 (Trial 2). x Column means with a letter in common are n P=0.05). 73 Figure 5 . Botrytis cinerea and treated with biorational products A: Untreated control, B: Bac illus subtilis (Serenade Opti), C: Streptomyces lydicus (Actinovate), D: Ulocladium oudemansii ( BotryStop), E: Pseudomonas chlororaphis (Zio), F: Aureobasidium pullulans ( Botector), G: Gliocladium catenulatum ( Prestop) , H: F enhexamid (Decree) G H E F C D A B 74 DISCUSSION Botrytis blight caus es a loss of millions of dollars each year (Steiger, 2007 ; Dean et al., 2012). In our study, a ll cultivars evaluted were susceptible but signifi cant differences were observed. e severity and were similar to as had less disease across all Some petunia cultivars have be en found to be resistant to B. cinerea (Krahl and Randle, 1999 ; Weddle, 1976 ) but are no longer available. Floryszak - wieczorek et al. (2007) reported that in resistant geranium cultivars, there was an early nitric oxide (NO) burst with subsequent secondary waves of NO, whereas in the susceptible cultivar, there was an overproduction of NO as the disease progressed but an early burst and seconda ry wave of NO was lacking. An early and high co ncentration of NO generates a strong signal for effective defense in resistant cultivars. T he accumulation of secondary metabolites may induce the host response to the pathogen as no qualitative resistance to B . cinerea has been found. Inconsistent result susceptible in Trial 1 but was among the least susceptible cultivars in Trial 3 . Easy Wave Red Improved susceptible in Trial 1 were moderately suscept ible in Trials 2 and 3 according to disease severity assessments . When Krahl and Randle (1999) evaluat ed 48 petunia cultivars for B. cinerea resistance, they observed variation among cultivars over two seasons but Pink Sensation Improved was consistent ly resistant . 75 Inconsistency has been reported for B. cinerea on lisianthus ( Wegulo and Vilchez, 2007) and geranium cultivars ( Uchneat et al.,1999) against B . cinerea . Biorationals could be used as an alternative or in conjunction with traditional fungicides to limit B .cinerea . Commercially available biorationals were tested for their ability to co ntrol that was mor e resistant than others included in our trial. M any of t he biorationals provided a similar level of control as the fungicide standard. Higher disease severity was observed in the Tri al 2 compare d to Trial 1. Among the tested products, Aureobasidium pullula ns ( Botector ) and Gliocladium catenulatum ( Prestop ) provided effective control similar to the fungicide standard in both trials with reduced disease severity and AUDPC values. A. pul lulans is a yeast that inhibits mycelial growth and conidial germination of B. cinerea through the production of diffusible and volatile inhibitory antifungal compounds (Yalage et al., 2020) and secretion of hydroly tic enzymes including chitinase, - 1,3 - glucanase , and protease ( Zhang et al. , 2010 ; Chen et al . , 2018 ) . It also acts as an indirect antagonist by suppressing B. cinerea by competing for space and nutrition ( Castoria, et al., 2001; Zhang et al., 2010). Our findings that A. pullulans effectively limited B . cinerea is supported by previous studi es where A. pullulans was moderate to highly effective for B . cinerea control on greenhouse tomato and cucumber and reduced the number of diseased fruits and stem lesions (Dik and Elad , 1999) . The efficacy of A. pullulans against B. cinerea has been reported for apples (Zhang et al., 2010), strawberry (Lima et al. , 1997 ; Sylla e t al., 2015) , and grapes (Fedele et al., 2020; Pertot et al., 2017) . When applied in combination with T. harzianum T39, A. pullulans significantly reduced stem lesions on tomat o compared to A. pullulans alone (Dik et al. , 1999) . 76 Suppression of Botrytis by G. catenulatum has been described through the a ntagonistic mechanism of antibiosis and mycoparasitism (Pal and Gardener, 2006; Jacometti et al., 2010) . G. catenulatum ( Prestop) similar to our study have successfully control the B. cinerea on greenhouse tomatoes (Utkhede and Mathur, 2006) and geranium (Elmhirst et al., 2011) . Other species of Gliocladium ( G. roseum ) effectively controlled Botrytis blight by suppressing spore production, reducing disease incidence on strawberry, raspberry and greenhouse flowers (begonia, cyclamen and geranium) and vegetables (cucumber, pepper and tomato) (Sutton et al., 1997) . In our study, Pseudomonas chlororaphis (Zio) resulted in disease severity ratings and AUDPC values similar to the fungicide standard fenhexamid for both trials: an excepti on was observed with AUDPC data for Trial 1. Pseudomonas spp . inhibits the conidial germination of B. cinerea by secreting volati le metabolites with fungistatic effects (Swadling and Jeffries, 1998 ; Redouan, et al., 2018) . Pseudomonas fluorescens effectively supressed B. cinerea sporulation and significantly reduced disease on greenhouse petunia (G ould et al., 1996 ) and tomato (Yildiz et al., 2007 ) . In vitro evaluation of P seudomonas spp. showed B. cinerea my celial growth inhibition of 65% with 100% radial growth inhibition through the production of votalite antifungal compounds (Redouan et al., 2018) . According to South et al. ( 2020) P. protegens AP54, P. chlororaphis 14B11 and P. fluorescens 89F1 effectively controlled B. cinerea in pet unia based on a disease severity index and AUDPC values. None of the products with Bacillus species; B. subtilis (Serenade Opti) , B. amyloliquefaciens (Serifel) and B. mycoides (LifeGard) , tested in this study reduced disease severity compared to the untr eated inoculated control, although B. mycoides ( LifeGard ) was similar to the standard fungicide fenhexamid ( Decre e ) . Bacillus species secrete antimicrobial compounds, antibiotics and lipopeptide - like compounds and acts directly as an antagonist 77 against B . cinerea hyphae (Salvatierra - Martinez et al., 2018) . I n contrast to our result, B .cinerea has been effectively suppressed in many other crops by B. subtilis (Abbey et al. , 2020; Elmhirst et al., 2011; Pertot et al., 2017) and B. amyloliquefaciens (Nakkeeran et al., 2020; Salvatierra - M a rtinez et al., 2018; Zhou et al., 2020) . Ruiz - Moyano et al. ( 2020 ) conducted in - vivo assays of strawberry and cherries and determined that Hanseniaspora spp. isolates provided increased efficacy and reduced B . cinerea mycelial growth and development: H. uvarum 793 was selected as potential bio rational. Combining biorational products with different mechanism could reduce performance variability and improve efficacy (Guetsky et al. , 2001; Guetsky et al., 2002) . Biorational products can be combined with fungicides to reduce the number of fungicide applications thereby reducing the risk of pathogen resistance and providing effective control of B. cinerea (Rotolo et al., 2018) . b others in our study. Growers interested in using biorationals may want to consider selecting cultivars that are less suceptible to B. cinerea . Gliocladium catenulatum ( Prestop ) , Aureobasidium pullulans ( Botector ) and Pseudomonas chlororaphis ( Zio ) effective ly lim ited disease in our trials when tested on a petunia cultivar determined to have a level of resistance to Botrytis blight. 78 LITERATURE CITED 79 LITERATURE CITED 1. Abbey, J. A., Percival, D., Asiedu, S. K., Prithiviraj, B., and Schilder, A . 2020. Management of Botrytis blossom blight in wild blueberries by biological control agents under field conditions. Crop Prot . 131 :105078. 2. Bertetti, D., Monchiero, M., Garibaldi , A., and Gullino, M. L. 2020. Monitoring activities on fungicide resistanc e in Botrytis cinerea carried out in vineyards in North - West Italy in 2018. J . Plant Dis . Prot ect . 127 : 123 127. 3. Castoria , R., De Curtis, F., Lima, G., Caputo, L., Pacifico, S., and De Cicco, V. 2001. Aureobasidium pullulans (LS - 30) an antagonist of postha rvest pathogens of fruits: Study on its modes of action. Postharvest Biol . Technol . 22:7 17. 4. Chen, P. H., Chen, R. Y., and Chou, J. Y. 2018. Screening and evaluation of yeast antagonists for biological control of Botrytis cinerea on strawberry fruits. My cobiology . 46 : 33 46. 5. Choudhary, D. K., and Johri, B. N. (2009). Interactions of Bacillus spp. and plants - With special reference to induced systemic resistance (ISR). Microbiol . Res . 164 : 493 513. 6. Dean , R ., Van Kan , J . A . L., Pretorius , Z . A ., Hammond - Ko sack , K . E ., Di Pietro , A ., Spanu , P . D ., Rudd , J . J ., Dickman , M ., Kahmann , R ., Ellis , J ., and Foster , G . D. 2012. The Top 10 fungal pathogens in molecular plant pathology. Mol . Plant Pathol . 13 : 414 430. 7. Dik, A. J., and Elad, Y. 1999. Comparison of antago nists of Botrytis cinerea in greenhouse - grown cucumber and tomato under differe nt climatic conditions. Eur . J . Plant Pathol . 105 : 123 137. 8. Dik, A. J., Koning, G., and Köhl, J. 1999. Evaluation of microbial antagonists for biological control of Botrytis cin erea stem infection in cucumber and tomato. Eur . J . Plant Pathol. 105:115 122. 9. Dik A . J ., and Wubben , J . P . 2004 Epidemiology of Botrytis cinerea diseases in greenhouses. In: Botrytis: biology, pathology, and control. Elad Y, Williamson B, Tudzynski P, De len N (eds) . Kluwer Academic Publishers, Dordrecht, pp 319 333 . 10. E lad, Y. 2016. Cultural and integrated control of Botrytis spp. In : Botrytis - The Fungus, the Pathogen, and its Management in Agricultural Systems . Fillinger , S. and Y. Elad (Eds.) : 149 164. 80 11. Elad, Y., and Shtienberg, D. 1995. Botrytis cinerea in greenhouse vegetables: chemical, cultural, physiological, and biological controls and their integration. Integr . Pest Manag . Rev . 1:15 29. 12. Elmhirst, J. F., Haselhan, C., and Punja, Z. K. 2011. Evalua tion of biological control agents for control of bot rytis blight of geranium and powdery mildew of rose. Can . J . Plant Pathol. 33:499 505. 13. Fedele, G., González - Domínguez, E., Ammour, M. S., Languasco, L., and Rossi, V. 2020. Reduction of Botrytis cinerea colonization of and sporulation on bunch trash. Plant Dis . 104 : 808 816. 14. Fernández - Ortuño, D., Grabke, A., Li, X., and Schnabel, G. 2015. Independent Emergence of Resistance to Seven Chemical Classes of Fungicides in Botrytis cinerea . Phytopathology . 105 : 4 24 432. 15. Floryszak - wieczorek, J., Arasimowicz, M., Milczarek, G., Jelen, H., and Jackowiak, H. 2007. Only an early nitric oxide burst and the following wave of secondary nitric oxide generation enhanced effective defen s e responses of Pelargonium to a necr o trophic pathogen . New Phytol. 175: 718 730. 16. Gould, A. B., Kobayashi, D. Y., and Bergen, M. S. 1996. Identification of bacteria for biological control of Botrytis cinerea on petunia using a petal disk assay. Plant Dis. 80:1029 - 1033. 17. Guetsky, R., Shtienberg , D., Elad, Y., and Dinoor, A. 2001. Combining biocontrol agents to reduce the variability of biological control. Phytopathology . 91 : 621 627. 18. Guetsky, Ruth, Shtienberg, D., Elad, Y., Fischer, E., and Dinoor, A. 2002. Improving biological control by combin ing biocontrol agents each with several mechanisms of disease suppression. Phytopathology . 92 : 976 985. 19. Hahn, M. 2014. The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study. J . Chem . Biol . 7 : 133 141. 20. Hausbeck, M. K. , and Moorman, G. W. 1996. Managing Botrytis in greenhouse - grown flower Crops. Plant Dis. 80:1212 - 1219. 21. Hausbeck, M. K., and Pennypacker, S. P. 1991. Influence of g rower a ctivity and d isease i ncidence on c oncentrations of a irborne c onidia of Brotytis cine rea a mong g eranium stock plants. Plant Dis . 75 : 798 803. 81 22. Hausbeck, M., Pennypacker, S., and Stevenson, R. 1996. The effect of plastic mulch and forced heated air on Botrytis cinerea on geranium stock plants in a research greenhouse. Plant Dis. 80:170 - 173. 23. Horst, L. E., Locke, J., Krause, C. R., McM ahon, R. W., Madden, L. V., and Hoitink, H. A. J. 2005. Suppression of Botrytis blight of begonia by Trichoderma hamatum 382 in peat and compost - amended potting mixes. Plant Dis. 89:1195 1200. 24. Hu, M. - J., Cox, K. D., and Schnabel, G. 2016. Resistance to Increasing Chemical Classes Botrytis cinerea . Phytopathology . 106 : 1513 1520. 25. Jacometti, M. A., Wratten, S. D., and Walter, M. 2010. Review: Alternatives to syntheti c fungicides for Botrytis cinerea management in vineyards. Australian Journal of Grape and Wine Research. 16(1):154 172. 26. Jarvis, W. R. 1989. Managing d iseases in g reenhouse c rops. Plant Dis. 73:190 - 194. 27. Jiang, C., Liao, M., Wang, H., Zheng, M., and Xu, J. 2018. Bacillus velezensis , a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea . Biol . Control. 126:147 157. 28. Krahl, K. H., and Randle, W. M. 1999. Resista nce of petunia phenotypes to Botrytis cine rea . HortScience. 34:690 692. 29. Lima, G., Ippolito, A., Nigro, F., and Salerno, M. 1997. Effectiveness of Aureobasidium pullulans and Candida oleophila against postharvest strawberry rots. Postharvest Biol . Technol . 10 : 169 178. 30. Moyano, C., Gómez, V., and M elgarejo, P. 2004. Resistance to pyrimethanil and other fungicides in Botrytis cinerea populations collected on vegetable crops in Spain. J . Phytopathol . 152 : 484 490. 31. Muñoz, M., Faust, J. E., and Schnabel, G. 2019. Characterization of Botrytis cinerea fr om commercial cut flower roses. Plant Dis . 103 : 1577 1583. 32. Nakkeeran, S., Priyanka, R., Rajamanickam, S., and Sivakumar, U. 2020. Bacillus amyloliquefaciens alters the diversity of volatile and non - volatile metabolites and induces the expression of defen s e genes for the management of Botrytis leaf blight of Lilium under protected conditions. J . Plant Pathol. 82 33. Olson, H. A., and Benson, D. M. 2 007. Induced systemic resistance and the role of binucleate Rhizoctonia and Trichoderma hamatum 382 in biocontrol of Botrytis blight in geranium. Biol . Control. 42:233 - 241. 34. Pal, K. K., and Gardener, B. M. 2006. Biological control of plant pathogens. The Plant Health Instructor. 2:1117 - 1142. 35. Paulitz, T. C., and Belanger, R. R. 2001. Biological control in greenhouse syst em. Annu . Rev . Phytopathol. 39:103 133. 36. Pertot, I., Giovannini, O., Ben anchi, M., Caffi, T., Rossi, V., and Mugnai, L. 2017. Combining biocontrol agents with different mechanisms of action in a strategy to control Botrytis cinerea on grapevine. Crop Prot . 97 : 85 93. 37. Punja, Z. K., and Utkhede, R. S. 2003. Using fungi and yeast s to manage vegetable crop diseases. Trends Biotechnol. 21:400 407. 38. Redouan, Q., Rachid, B., Abedrahim, A., Hassan, M. EL, and Bouchra, C. 2018. E ffectiveness of beneficial bacteria P seudomonas spp. to control grey and green mold. In PP: 933 939 . 39. Rotolo, C., De Miccolis Angelini, R. M., Dongiovanni, C., Pollastro, S., Fumarola, G., Di Carolo, M., Perrelli, D., Na tale, P., and Faretra, F. 2018. Use of biocontrol a gents and botanicals in integrated management of Botrytis cinerea in table grape vineyards. Pest Manag . Sci . 74 : 715 725. 40. Ruiz - Moyano, S., Hernández, A., Galvan, A. I., Córdoba, M. G., Casquete, R., Serrad illa, M. J., and Martín, A. 2020. Selection and app lication of antifungal VOCs - producing yeasts as biocontrol agents of grey mould in fruits. Food Microbio l . 92 : 103556 . 41. Saito, S., Michailides, T. J., and Xiao, C. L. 2019. Fungicide - resistant phenotypes in Botrytis cinerea populations and their impact on c ontrol of gray mold on stored table grapes in California. Eur . J . Plant Pathol . 154 : 203 213. 42. Salvatierra - Martinez, R., Arancibia, W., Araya, M., Aguilera, S., Olalde, V., Bravo, J., and Stoll, A. 2018. Co lonization ability as an indicator of enhanced biocontrol capacity An example using two Bacillus amyloliquefaciens strains and Botrytis cinerea infection of tomatoes. J . Phytopathol . 166 : 601 612. 43. Samarakoon, U., Bennett, K., Jent, J., Chiu, C., and Sch, G . 2016. Alternative compounds to control gray mold . In: Petunia flower meltdown . Grower Talks . 83 44. Samarakoon, U. C. , Schnabel, G., F aust, J. E., Bennett, K., Jent, J., Hu, M. J., Basnagala, S., and Williamson , M. 2017. First report of resistance to multiple chemical classes of fungicides in B otrytis cinerea , the causal agent of gray mold from greenhouse - grown petunia in F lorida . Plant Dis. 101:1052. 45. Simko, I., and Piepho, H. P. 2012. The area u nder the disease progress stairs: Calculation, advantage, and app lication. Phytopathology . 102 : 381 389. 46. South, K. A., Peduto Hand, F., and Jones, M. L. 2020. Beneficial b acteria i dentified for the control of Botrytis cinerea in petunia greenhouse production. Plant Dis. 104:1801 1810. 47. Steiger, D. 2007. Global ecoonomic importance of Botrytis protection. In : 14 th I nternational B otrytis S ymposium. African Sun MeDIA Pty (Ltd.). Page 7. 48. Sutton, J. C., Li, D., Peng, G., Yu, H., Z hang, P., and Valdebenito - Sanhueza, R. M. 1997 . Gliocladium roseum : A versatile adversary of Bot rytis cinerea in crops antagonistic. Plant Dis . 81 : 316 328. 49. Swadling, I. R., and Jeffries, P. 1998. Antagonistic properties of two bacterial biocontrol agents of grey mould disease. Biocontrol Sci . Technol. 8:439 448. 50. Sylla, J., Alsanius, B. W., Krüger, E., and Wohanka, W. 2015. Control of Botrytis cinerea in strawberries by biological control agents applied as single or combined treatments. Eur . J . Plant Pathol . 143 : 461 471. 51. Uchneat, M. S., Zhigilei, A., and Craig, R. 1999. differential response to foli ar infection with Botrytis cinerea within the genus P elargonium . J. Am. Soc. Hortic. Sci. 124:76 80. 52. USDA - National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. Retrieved from: https://www.nass.usda.gov/Publications/Todays_Report s/reports/floran19.pdf 53. Utkhede, R. S., and Mathur, S . 2006. Preventive and curative biological treatments for control of Botrytis cinerea stem canker of greenhouse tomatoes. Bio l . Control . 51 : 363 373. 54. Weddle, C.L. 1976. Petunias. In: Bedding plants: A ma nual on the culture of bedding plants as a greenhouse crop. Mastalerz , J.W. (ed.). Penn. Flower Growers, University Park : 252 270 55. Wegulo, S. N., and Vilchez, M. 2007. Evaluation of l isianthus c ultivars for r esistance to Botrytis cinerea . Plant Dis. 91:997 1001. 84 56. Williamson, B., Tudzynski, B., Tudzynski, P., and Van Kan, J. A. L. 2007. Botrytis cinerea : The cause of grey mould disease. Mol . Plant Pathol. 8:561 580. 57. Yalage Don, S. M., Schmidtke, L. M., Gambetta, J. M., and Steel, C. C. 2020. Aureobasidium pul lulans volatilome identified by a novel, quantitative approach employing SPME - GC - MS, suppressed Botrytis cinerea and Alternaria alternata in vitro. Sci . Rep . 10 : 1 13. 58. and Türküsay, H. 2007. The effects of biolo gical and chemical treatment on gray mold disease in tomatoes grown under greenhouse conditions. Tur k. J . Agric . For. 31:319 325. 59. Zhang, D., Spadaro, D., Garibaldi, A., and Gullino, M. L. 2010. Efficacy of the antagonist Aureobasidium pul lulans PL5 agains t postharvest pathogens of peach, apple and plum and its modes of action. Biol . Control. 54:172 180. 60. Zhou, Q., Fu, M., Xu, M., Chen, X., Qiu, J., Wang, F., Yan, R., Wang, J., Zhao, S., Xin, X., and Chen, L. 2020. Application of antagonist Bacillus amyloli quefaciens NCPSJ7 against Botrytis cinerea in postharvest Red Globe grapes. Food Sci . Nutr. 8:1499 1508.