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Canadian Journal of Plant Pathology

ISSN: 0706-0661 (Print) 1715-2992 (Online) Journal homepage: https://www.tandfonline.com/loi/tcjp20

Biological control of powdery mildew on Cornusflorida using endophytic Bacillus thuringiensis

Emily RotichEMILY ROTICH, Margaret T. MmbagaMARGARET T. MMBAGA &Jacqueline JoshuaJACQUELINE JOSHUA

To cite this article: Emily RotichEMILY ROTICH, Margaret T. MmbagaMARGARET T. MMBAGA& Jacqueline JoshuaJACQUELINE JOSHUA (2019): Biological control of powdery mildew onCornus�florida using endophytic Bacillus�thuringiensis, Canadian Journal of Plant Pathology, DOI:10.1080/07060661.2019.1641555

To link to this article: https://doi.org/10.1080/07060661.2019.1641555

Accepted author version posted online: 08Jul 2019.Published online: 31 Jul 2019.

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Disease control/Moyens de lutte

Biological control of powdery mildew on Cornus florida usingendophytic Bacillus thuringiensis

EMILY ROTICH1, MARGARET T. MMBAGA2 AND JACQUELINE JOSHUA3

1College of Sciences and Mathematics, Department of Biology, Belmont University, Nashville, TN 37212, USA2College of Agriculture, Human and Natural Sciences, Department of Agricultural and Environmental Sciences, Tennessee State University,Nashville, TN 37209, USA3Division of Crop Science, Monsanto Company, Chesterfield, MO, 63017, USA

(Accepted 4 July 2019)

Abstract: Powdery mildew (Erysiphe pulchra) is a devastating disease in nursery production of flowering dogwood (Cornus florida L.).Disease management relies heavily on season-long use of chemical fungicides and there is a demand for alternative strategies. An endophyticbacterium IMC8 was isolated from disease-free C. florida stem tissue, and was shown to be an effective biological control agent againstE. pulchra in growth chamber, greenhouse and shadehouse environments. This isolate, identified as Bacillus thuringiensis, exhibited normalgrowth in NaCl concentrations up to 6%, pH 5 to 11, and temperatures up to 50°C. It was compatible with the conventional fungicidethiophanate methyl and clarified hydrophobic extract of neem seed oil at label recommended concentrations. The isolate IMC8 producedvolatile compounds identified by GC/MS, which included antifungal and antibacterial compounds and suggested antibiosis as a mode ofaction. Scanning electron microscopy revealed lysis of powdery mildew spores and hyphae, which suggested parasitism as a second mode ofaction. These observations suggested that the isolate IMC8 has potential as a biological control agent in the management of powdery mildewon C. florida, and would tolerate a wide range of salinity, pH and high temperature conditions. It could be included as a component ofintegrated pest management in rotation or tank mix with lower levels of conventional fungicides, or used with clarified hydrophobic extractof neem seed oil.

Keywords: biological control agents, endophytes, Erysiphe pulchra, integrated disease management, mycoparasitism

Résumé: L’oïdium (Erysiphe pulchra) est une maladie dévastatrice dans la production en pépinière de cornouiller à fleurs de Floride (Cornusflorida). La gestion des maladies repose largement sur l’utilisation de produits chimiques pendant toute la saison. Couramment, il existe unedemande pour des stratégies alternatives au produit chimique. La recherche d’agents de lutte biologique contre l’oïdium a permis l’isolementd’une bactérie endophyte, IMC8, à partir de tissu de C. florida indemne de maladie. L’isolat IMC8 s’est avéré être un agent de luttebiologique efficace contre E. pulchra dans les environnements de chambre de culture, de serre et serre d’ombre. Cet isolat identifié commeBacillus thuringiensis a présenté une croissance normale dans une large gamme de niveaux de salinité et de pH, allant jusqu’à 6% de NaCl etpH entre 5 et 11, ainsi qu’à des températures allant jusqu’à 50°C. Il était compatible avec le fongicide classique thiophanate de méthyle et lebiofongicide, l’extrait hydrophobe clarifié d’huile de graines de neem, à des concentrations couramment utilisées dans les applications delutte contre l’oïdium au pepinere commercial. L’isolat IMC8 a produit des composés volatils identifiés par GC/MS, parmi lesquels descomposés antifongiques et antibactériens ont suggéré l’antibiose comme mode d’action. D’ailleurs, la microscopie électronique à balayagea révélé la lyse des spores et des hyphes de l’oïdium, suggérant le parasitisme comme second mode d’action. Ces observations suggèrent quel’isolat IMC8 présente un potentiel en tant qu’agent de lutte biologique dans la gestion de l’oïdium et qu’il tolérerait une large gamme deconditions salines, de pH et de températures élevées. Les résultats présentés suggèrent que l’isolat IMC8 peut constituer une alternative auxfongicides classiques pour la gestion de l’oïdium et réduire l’utilisation de fongicides dans la production en pépinière de C. florida, soit

Correspondence to: Emily Rotich. E-mail: emily.rotich@belmont.edu

Can. J. Plant Pathol., 2019https://doi.org/10.1080/07060661.2019.1641555

© 2019 The Canadian Phytopathological Society

Published online 31 Jul 2019

comme composante de la lutte intégrée en alternance, soit en mélange en réservoir de pulvérization avec des niveaux inférieurs de fongicidesclassiques, soit il peut être utilisé avec un extrait hydrophobe clarifié d’huile de graines de neem.

Mots clés: agents de lutte biologique, endophytes, Erisyphe pulchra, la lutte intégrée contre des maladies des plantes, mycoparasitisme

Introduction

Cornus florida (flowering dogwood) is a popular orna-mental tree throughout the southeastern United States.Powdery mildew caused by Erysiphe (Sect.Microsphaera) pulchra (Cook & Peck, Braun &Takamatsu), is a devastating disease of C. florida(Hagan and Mullen 1995; Mmbaga 1998, 2000; Liet al. 2009). The white, powdery appearance caused bydust-like mycelium on leaf surfaces reduces the aestheticvalue of infected trees, as well as the rate of photosynth-esis, causing stunted growth and increasing the timeneeded for plants to reach optimal size for plant sale(Windham 1996; Mmbaga and Sauvé 2004; Agrios2005; Shi et al. 2008). Severe infection with powderymildew has also been shown to reduce flower set and theamount of fruit formed (Heald 1999).Since the emergence of powdery mildew on flowering

dogwood in the early 1990s, management strategies haverelied heavily on conventional fungicides with applica-tions starting soon after bud-break and continuing allseason-long (Windham 1994; Mmbaga 2000).Fungicide applications such as wettable sulfur primarilyare used preventatively, but they are not effective againstexisting infections (Ash 1999). However, other fungi-cides including thiophanate methyl and propiconazoleare commonly used all season long as protective aswell as curative remedies. The season-long use of che-mical fungicides has significantly increased the cost ofproduction, leading to abandonment of C. florida pro-duction by small-scale growers in favor of other orna-mentals (Li et al. 2009). In addition, there are concernsover accidental exposure of humans and wildlife to thesechemicals, environmental contamination, adverse effectson microbial biodiversity, and beneficial non-target nat-ural microflora that otherwise protect plants against otherpathogens (de Jager et al. 1995; Sharma et al. 2011).Continued use of fungicides also contributes to thepotential for development of resistance in pathogens(Sharma et al. 2011). There is a demand for safer, non-chemical alternatives for powdery mildew management.Although genetic disease resistance is an environmen-

tally safe and affordable approach for disease manage-ment, it takes a long time to develop resistant genotypes,especially on tree crops. Flowering dogwood seedlingsrequire four years to initiate flowering and then produce

few flowers for breeding purposes. In addition, break-down of resistance to powdery mildew or environmentaleffects which render the resistance ineffective at differ-ent locations is a problem (Mmbaga and Sauvé 2004).A few cultivars including ‘Cherokee Brave’, ‘Karen’sAppalachian Blush’, ‘Jean’s Appalachian Snow’,‘Kay’s Appalachian Mist’, and ‘Appalachian Joy’ havesome degree of resistance to powdery mildew, but nonehas a high level of resistance. An integrated diseasemanagement system that combines different methodswould be ideal to manage powdery mildew infections(Witte et al. 2000; Li et al. 2009).The use of biological and biorational products to sup-

press fungal pathogens has been recommended in horticul-tural crop production due to their lower level of toxicity(Stimmel 1996). There are a few biorational products thatare labeled for powdery mildew control and include plantextracts from neem oil marketed as Triact 70, and neemGold as well as potassium bicarbonate salts marketed asArmicarb and Kaligreen (Mmbaga and Sheng 2002). Theincorporation of Armicarb and Triact 70 in fungicide rota-tions has been reported to reduce conventional fungicideusage by up to 56% (Mmbaga and Sheng 2002). Biologicalcontrol strategies which use one living organism to controlthe growth and proliferation of another are safer and inex-pensive alternatives to conventional agrochemicals (VanDriesche and Bellow 1996; Hawley and Eitzen 2001;Gardener and Fravel 2002; Kiss 2004; Sharma et al.2011). Biological-based disease management can be inte-grated with other strategies such as host resistance and lowlevels of fungicides. Since bio-control agents are livingorganisms, it is important to investigate the conditionsnecessary for their optimal development, survival and effi-cacy (Bélanger and Labbé 2002). Screening of diverseepiphytes for biological control agents for dogwood pow-dery mildew identified bacteria, fungi and yeasts that sup-pressed powdery mildew (Mmbaga et al. 2008, 2016;Mmbaga and Sauvé 2009). Screening of diverse endo-phytes identified the bacterial isolate IMC8 to be effectivein suppressing powdery mildew (E. pulchra) in floweringdogwoods. This study focused on the evaluation of theefficacy of isolate IMC8 in limiting powdery mildew dis-ease severity, its identification and characterization. Thestudy also evaluated this isolate in different environmentalconditions and assessed its potential mode of action inpowdery mildew control.

E. Rotich et al. 2

Materials and methods

Evaluation of the impact of isolate IMC8 on powderymildew disease severity

The effect of IMC8 on the severity of powdery mildewon flowering dogwood was evaluated using (i)a detached leaf technique in a moist chamber set at23 ± 3°C and 99–100% relative humidity, (ii) 3-month-old C. florida seedlings grown in a greenhouse environ-ment at 26–28°C temperature in McMinnville (NurseryResearch Center) and in Nashville’s University ResearchFacility, and (iii) two year old seedlings of three geno-types ‘Cherokee Princess’ (susceptible), ‘R14ʹ and ‘MI9ʹ(moderately resistant) grown in a shadehouse environ-ment under 50% shadecloth. For the detached leaf tech-nique, leaves of similar size were harvested from diseasefree C. florida “Cherokee Princess” and surface-sterilized with 10% alcohol for 60s, rinsed twice insterilized water, and blotted dry using heat sterilizedtissue paper. The leaves were then placed in a moistchamber lined with a double layer of sterilized tissuepaper kept moist using sterilized water. The endophyticbacterium IMC8 previously isolated from flowering dog-wood stems, and maintained in nutrient agar (NA) orLuria-Bertani (LB) agar at −80°C was retrieved as aninoculum source. The inoculum suspension of approxi-mately 106 CFU/mL was prepared from 36-h-old cul-tures by the plating technique and inoculumconcentrations were assessed using previously standar-dized odometer readings corresponding to 106 CFU/mL.Detached leaves were spray inoculated using sterilizedhand-held atomizers. Non-treated controls were sprayedwith sterilized water. A replication of four leaves pertreatment arranged in a randomized complete blockdesign was used. At two hours after inoculation withIMC8, the detached leaves were brush inoculated withpowdery mildew spores from previously infected leavesand incubated at 23 ± 3°C. Disease severity was assessed14 d after inoculation using a modified Horsfall andBarratt (1945) rating scale of 0 to 5, where 0 = nodisease symptoms; 1 = 1–10%; 2 = 11–25%;3 = 26–50%; 4 = 51–75%; and 5 = 76–100% of leaveswith disease symptoms. The experiment was performedtwice and the two years’ data were pooled.The effect of IMC8 on powdery mildew was also

evaluated in greenhouse experiments using 3-month-oldC. florida seedlings and in shadehouse experimentsunder 50% shadecloth using 2-year-old grafted plantsof three genotypes ‘Cherokee Princess’, ‘R14ʹ and‘MI9ʹ grown in 3.78 L containers. The fungicide thio-phanate methyl (Cleary’s 3336F, Cleary Chemicals,

Dayton, NJ) and water were used as the positive andnegative controls, respectively. Treatments with isolateIMC8 were initiated as soon as the initial symptoms ofpowdery mildew were observed in the local area in earlyto mid-May. The experiments were arranged ina randomized complete block design with a replicationof 40 individual plants per treatment in greenhouseexperiments and eight individual plants per treatment inshadehouse experiments. Previously infected plants ran-domly placed in greenhouse and shadehouse experimen-tal areas provided a continuous source of air-bornepowdery mildew inoculum. Plants were sprayed withtreatments to run-off every 7–10 days, and treatmentapplications were terminated at the end of August, coin-ciding with the timing of grower practices in powderymildew spray programs. Disease severity was assessedas described above. Data were analyzed using StatisticalAnalysis System (SAS) software v. 9.4 (SAS instituteInc., Cary, NC), with t-tests to compare means followingPROC ANOVA and PROC GLM. Least significant dif-ferences (LSD) were calculated at P ≤ 0.05 (Ott andLongnecker 2001).

Identification and characterization of isolate IMC8

The bacterial isolate was identified using molecularsequence analysis, as well as morphological, and phy-siological characteristics based on colony shape and size(Janda and Abbott 2002). Bacterial DNA was extractedfrom the isolate using a DNA blood and tissue kit(Qiagen Inc, Valencia, CA) following the manufacturer’srecommendations (DNeasy Blood and tissue kitHandbook, July 2006). The DNA concentration andrelative purity was determined using a Nanodrop Lite(Thermo Fisher Scientific, Wilmington, DE). A pair ofoligonucleotide primers (RW01: 5ʹ-AAC TGG AGGAAG GTG GGG AT-3ʹand DG74: 5ʹ-AGG AGG TGATCC AAC CGC A-3ʹ) universal for bacteria (Greisenet al. 1994) were used to amplify a 370 bp region of the16S rRNA gene. PCR amplification was carried out ina final volume of 25 μL with PCR buffer 1X (Promega,Madison, WI), 2.5 mM MgCl2, 200 μM of each dNTP,100 pM of each primer, 20 ng of genomic DNA and 2.5units of Taq polymerase (Promega, Madison, WI).Amplification was carried out in a PTC 100 ThermalCycler® (Boier Lifepro, Grand Island, NY) programmedwith an initial denaturation temperature of 95°C for5 min, followed by 34 cycles consisting of denaturationat 95°C for 1 min, annealing of the primers at 55°C for1 min, and a final extension step at 72°C for 10 min.

Biological control of powdery mildew on Cornus florida 3

The PCR product was purified using an Exosap DNApurification kit (Invitrogen, Life Technologies, Inc.Carlsbad, CA) following the manufacturer’s recom-mended protocols. The PCR products sequenced atDavis Sequencing Inc. (Davis, CA). A BLAST search(Altschul et al. 1997) was performed with GenBank(ncbi.nlm.nih.gov/genbank/) sequence data libraries,and the closest similarity match with other 16S rDNAgene sequences previously deposited in GenBank wasused to determine the identity of the bacterium. TheDNA sequence was deposited in GenBank (accessionno. MK245974).

Effect of different environmental conditions on thebacterial growth

The isolate was grown on nutrient broth (Difco BectonDickinson, Sparks, MD), adjusted to 106 CFU/mL andthen serially diluted six-fold before plating to determineits growth at different levels of salinity and pH, and inmedia amended with fungicides that are commonly usedin powdery mildew control. At the end of each study,colonies were counted to determine the number of col-ony forming units (CFU) at each condition. Theseexperiments used four replicates and each study wasperformed twice; the data were pooled for analysis.Log10 numbers of CFU/mL were analyzed using SAS.To evaluate the effect of temperature, the bacterial

isolate was streaked onto nutrient agar (NA) Petri plates,and incubated at 4, 23, 28, 37, 45 and 50°C to determinegrowth and morphological characteristics at the 24 h-oldculture stage. Growth was monitored for 72 h, andscored as: – no growth, + slight growth with up to 102

CFU/mL, ++ moderate growth (103 to 106 CFU/mL),and +++ good growth (with more than 106 CFU/mL).To determine the survival of the bacterial isolate in

different concentrations of sodium chloride (NaCl), thebacterium was grown in nutrient broth for 24 h andstreaked on NA plates containing 2, 4, 6, 8 and 10%w/v NaCl with 6 plates per salinity level. All plates wereincubated at room temperature for 48 h and bacterialgrowth was quantified using the previously developedgrowth curve of CFU/mL in serially diluted plates. Thebacterial isolate was also plated on NA with the pHadjusted to 5, 7, 9, 11, and 12 using hydrochloric acidand sodium hydroxide. The number of colonies wasquantified and expressed as CFU/mL.Fungicide tolerance was evaluated to determine com-

patibility of IMC8 with conventional fungicides and twobiorational products labeled for integrated managementof powdery mildew in C. florida. The fungicide

thiophanate methyl (Cleary 3336 F) and biorational pro-ducts, potassium bicarbonate (Armicarb100) and clari-fied hydrophobic extract of neem oil (Triact 70) Dhingraand Sinclair 1995) were previously tested using slightvariations of label recommended rates. The most effica-cious rates that did not harm dogwood plants wereselected for this study (Mmbaga and Sheng 2002).Application rates of thiophanate methyl were 0.78 mL/L, and potassium bicarbonate and neem oil extract wereat 6.6 g/L. These fungicides were dissolved in sterilizedmilli Q water and mixed into the NA just before pouringthe agar into the Petri plates. The CFU/mL was assessedup to a 72 h growth period.

Potential mode of action of IMC8

This study evaluated the possible involvement of (i)volatile compounds produced by the endophyte and (ii)parasitism of the powdery mildew fungus by IMC8isolate.Gas chromatography mass spectrometry (GC/MS) was

used to detect volatile compounds from IMC8. For thisanalysis, a single colony of the isolate was grown onnutrient agar for 48 h in glass vials covered with a poly-propylene hole cap top (Supelco, Bellefonte, PA) tomaximize the trapping of volatiles. The vials pre-concentrated the volatiles and allowed identificationand quantification of even small amounts, which weredetected through the solid phase microextraction(SPME) method and analyzed by GC/MS. The analysiswas performed using a Varian 3800 gas chromatographequipped with an ion-trap mass spectrometer VarianSaturn 2000 (Agilent technologies, Walnut Creek CA).The solid phase micro-extraction (SPME) fiber 50/30 μmDVB/Carboxen/PDMS StableFlex for manual holder(Supelco, Bellefonte, PA) was conditioned by runningit through the programmed cycle before exposing it tothe samples. The fiber was exposed into the headspace ofthe vial for 10 min, retracted, and then introduced intoa GC injector where the trapped volatiles were desorbedfrom the fiber to a chromatography column, usingHelium as a carrier. The injector temperature was set at220°C, and detector temperature at 265°C. Desorption inthe hot injector was programmed with an initial tempera-ture of 28°C held for 1 min, and then gradually increasedto 180°C, with a total run time of 40 min. The volatilesecondary metabolites released were identified using thechromatograph’s library search by comparing with thedatabase in the National Institute of Standards andTechnology (www.nist.gov).

E. Rotich et al. 4

While previous reports provided evidence that endo-phytes synthesize many secondary metabolites, somemetabolites produced may be different in the presenceof a pathogen (Tan and Zou 2001). However, as anobligate parasite, E. pulchra cannot be grown on artifi-cial medium. Thus, to determine if isolate IMC8 pro-duced different volatiles in the presence of a pathogen,this study used a facultative root rot pathogen witha broad host range, Macrophomina phaseolina. Thispathogen, the causal agent of charcoal rot disease, affecta diversity of crops including dogwood (Smith and Bega1964; Seymour 1969; Mmbaga et al. 2018) and IMC8has displayed efficacy against this pathogen in a differentstudy (Joshua and Mmbaga, unpublished). The studycomprised three treatments with three vials containing(i) a 5 mm plug of IMC8 alone from a 24 h-old culture,(ii) a 5 mm mycelial plug of M. phaseolina from 5 day-old cultures and a 5 mm plug of IMC8 endophyte andpathogen, and (iii) a 5 mm plug of M. phaseolina alone.The tests were done in duplicate and repeated once. Allsamples were incubated at 28°C for 48 h prior to con-ducting the sample analysis using GC/MS.The possible parasitism of the powdery mildew fun-

gus by IMC8 was examined using the detached leaftechnique in moist chambers in which powdery mildewinfected leaves of similar size, age and physical appear-ance were collected from a C. florida tree. The leaveswere placed in a moist chamber lined with a double layerof sterilized tissue paper kept moist using sterilizedwater. The detached leaves were spray inoculated withan IMC8 inoculum suspension of approximately 106

CFU/mL prepared from 36 h-old cultures using sterilizedhand-held atomizers. Non-treated controls were sprayedwith sterilized water. Five treatments represented time-points when leaves were harvested after inoculation withIMC8 and then fixed using Formalin–acetic acid–alcohol(FAA) overnight and stored at 4°C until processing forscanning electron microscopy (SEM). The time-pointsincluded 3 h, 6 h, 15 h, 48 h and 5 days post-inoculation and each time-point was replicated withfour leaves; the study was repeated once.

Leaf dehydration process for SEM

The samples were dehydrated in 30% ethanol for 15 min,50% ethanol for 15 min, 75% ethanol for 15 min, 85%ethanol for 15 min, 95% ethanol for 15 min, followed bypure ethanol three times for 15 min. Following the step-wise dehydration, samples were critical point dried inCO2 (Samdri®-PVT-3D, Tousimis, Rockville, MD),mounted onto sample stubs, and sputter coated with

gold-palladium (Cressington sputter coater, Ted PellaInc, Redding, CA) for 90 seconds. Following conductivesilver coating (Electron Microscopy Sciences, Hatfield,PA), the samples were subjected to SEM (Quanta 250Environmental Scanning Electron Microscope, HitachiS4200, Hillsboro, Oregon) to view the interaction ofIMC8 with the fungal hyphae and spores.

Results and discussion

Powdery mildew development is highly influenced byweather conditions. The growth chamber environmenthad a moderate temperature and high relative humidityknown to favor the disease (Agrios 2005). The isolateIMC8 was applied before powdery mildew was intro-duced and it was highly effective in controlling thedisease, with only traces of powdery mildew observedon the leaf surface. In the water control treatment, how-ever, powdery mildew covered 100% of the leaf sur-faces. This observation suggests that the moist chamberenvironment is highly favorable to IMC8 proliferation aswell as to the powdery mildew pathogen.Results from the greenhouse experiments are pre-

sented in Figs 1-2. The first experiment in Nashvillehad a higher disease severity and IMC8 was not effective(Fig. 1). In the second experiment in Nashville, powderymildew severity was high, IMC8 reduced powdery mil-dew but its effect was not statistically different fromwater control (Fig. 1). All three of the host genotypesevaluated developed moderate disease severity with nostatistical differences between them. However, therewere significant differences between treatments in allthree genotypes. Treatment with the fungicide wasmost effective in controlling powdery mildew followedby IMC8 as a biological control agent; the highest pow-dery mildew severity was observed on the water control(Fig. 2). The isolate IMC8 provided significant control ofpowdery mildew compared with the water controls, butit was less effective than the conventional fungicide.These results suggest that IMC8 may be used as part ofan integrated disease management plan that couldinclude moderately resistant cultivars or low levels ofcompatible fungicides.Based on the molecular tests, isolate IMC8 shared

99% similarity with Bacillus sp. (B. thuringiensis(NR_102506.1), B. cereus (NR_074540.1) andB. anthracis (NR_074453.1). The three species havethe same cell size and morphology and form oval spores.Further tests were conducted to differentiate the threeBacillus species biochemically: (i) the motility of thebacterial cells was visualized microscopically at 1000x

Biological control of powdery mildew on Cornus florida 5

using cultures grown in nutrient broth maintained at 30°C in a shaker set at 125 rpm for 3 h. Results werepositive for motility; (ii) growth on nutrient agar supple-mented with 5% sheep red blood cells showeda hemolysis pattern. (iii) An additional test for growthin 0.5 IU penicillin revealed positive results for penicil-lin resistance and negative for the ‘String of Pearls’reaction, which is the impairment of the cell wall thatBacillus anthracis exhibits in the presence of as little as0.05 IU penicillin (Chamberlain 2015). The three testseliminated the possibility of B. anthracis. To distinguish

B. thuringiensis from B. cereus, the IMC8 bacteria werestained for the detection of parasporal crystals associatedwith B. thuringiensis (Ejiofor and Johnson 2002). Thetest showed that IMC8 produces intracellular parasporalcrystals in association with spore formation. This testeliminated B. cereus and confirmed that isolate IMC8is B. thuringiensis (Fig. 3). The isolate formed endo-spores, an important feature for the microbial survivalin stressful environments (Reva et al. 2004), and whichfacilitates long-term storage and product formulation.Abundant colonies of the bacterium developed at

a wide range of temperatures from 23°C to 50°C withan optimum temperature of 28°C. These observationssuggest that IMC8 has potential application in agricul-tural production at a wide range of temperatures. Growthof IMC8 was reduced significantly starting at 4% NaClw/v and was further reduced at 6% compared with thecontrol; its growth was completely inhibited above 8%NaCl w/v. High soil salinity is an abiotic stress that oftenlimits crop production in affected areas. Bacillus specieshave been reported to be growth promoting (data notpresented) and also have been found to enhance planttolerance to abiotic stresses, leading to improved plantgrowth and production (Yang et al. 2009). Arid regionsare especially impacted by high soil salinity and biolo-gical control agents may be useful in increasing planttolerance to high soil salinity. Soil salinity levels affect-ing plant growth have an electrical conductivity (EC) of> 4 dS/m which corresponds to approximately 2.3%NaCl (U.S. Salinity Laboratory Staff 1954). In thisstudy, the bacterial isolate was able to grow at 4%NaCl and up to 6% NaCl, indicating an ability to survivein high saline conditions. There is a need to further

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Fig. 1 (Colour online) Powdery mildew severity on Cornus florida seedlings after foliar application of the bacterial biocontrol agent IMC8as compared with the fungicide thiophanate methyl and a water control under greenhouse conditions at two locations (McMinnville andNashville). Similar results were obtained in McMinnville Experiment 2 (not shown). Means within a group followed by the same letter arenot significantly different at p ≥ 0.05.

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Fig. 2 (Colour online) Powdery mildew severity on three Cornusflorida genotypes, including the susceptible cultivar ‘CherokeePrincess’ (CP) and two moderately resistant selections MI9 andR14, grown in a greenhouse at 26–28°C in Nashville, TN, and treatedwith foliar sprays of the bacterial isolate IMC8, the fungicide thio-phanate methyl or water (control) every 14 d over a 3 month growingseason. Treatment means within a group followed by the same lettersare not significantly different at p ≥ 0.05.

E. Rotich et al. 6

explore its potential supplementary role in improvingplant tolerance to high salinity.The isolate was able to grow at different pH levels

ranging from 5 to11, with a significant reduction ingrowth at pH 12. A neutral pH favors growth of mostorganisms and increased pH may impact plant growth(Margesin and Schinner 2001). The current results showa potential role of the bacterial isolate in helping to offsetthe impact of low or high pH on plant growth, butadditonal studies are needed. Previous studies reportedby Yang et al. (2009) showed that some bacteria helpplants tolerate abiotic stress.The results show that the growth of isolate IMC8

increased significantly in the presence of thiophanatemethyl (p < 0.05) compared with the control. Thisstrongly suggests that the isolate and thiophanate methylcan be used together, or in rotation as part of an integratedpowdery mildew management strategy. To our knowl-edge, this is the first report on such BCA/fungicide com-patibility. However, growth of the isolate was suppressedcompletely by the presence of potassium bicarbonate, butnot by neem oil extracts, both of which are commercialbiorational products. These results suggest that isolateIMC8 can be used in combination with thiophanatemethyl or clarified hydrophobic extract of neem oil toreduce fungicide use in dogwood nursery production,but not in combination or in rotation with potassiumbicarbonate. Mmbaga and Sheng (2002) reported 56%potential reduction in conventional fungicide usage byincorporating Armicarb or Triact70 in fungicide rotationsas part of integrated disease management.The GC-MS spectra showed various chromatographs

signifying different secondary metabolites produced by

isolate IMC8, and also from a combination of IMC8 andthe pathogen M. phaseolina (Table 1). The volatile com-pounds produced by IMC8 included cyclopentasiloxanedecamethyl, an active antimicrobial compound (Baishyaet al. 2018). However, most of the antifungal compoundswere produced in the presence of a pathogen (Table 1),including cycloheptasiloxane tetradecamethyl, cyclooc-tasiloxane hexadecamethyl, and heptadecane, 2,6,10,14-tetramethyl, which are known antimicrobials (Barakat2011; Keskin et al. 2012; Moustafa et al. 2013; Jasimet al. 2015). Mackie and Wheatley (1999) reported that

Fig. 3 (Colour online) Presence of crystal proteins in bacterial cells of isolates IMC8 treated with a differential crystal stain. (A) Lightmicroscopy image of a buffalo black-stained spore-crystal mixture consistent with Bacillus thuringiensis. Vegetative cells are stained (redarrow), black structures are crystal proteins (black arrow).

Table 1. Volatile compounds emitted by the IMC8 isolate alone orin the presence of Macrophomina phaseolina (MP) as identifiedby GC/MS SPME.

Sourcea Compound Functionb

Growth medium Control N/AFungicide Methoxy-phenyl-

oxime(thiophanatemethyl)

Antifungal

IMC8 Cyclopentasiloxanedecamethyl

Antimicrobial compound(Keskin et al. 2012; Baishyaet al. 2018)

IMC8 + MP Cycloheptasiloxanetetradecamethyl

Antimicrobial (Barakat 2011;Moustafa et al. 2013)

Cyclooctasiloxane,hexadecamethyl

Antimicrobial (Jasim et al.2015)

Heptadecane,2,6,10,14-tetramethyl

Antimicrobial (Manilal andIdhayadhulla 2014)

aRefers to the source of the compound identified.bRefers to the putative function of the compound.

Biological control of powdery mildew on Cornus florida 7

the response of fungi to bacterial volatile compounds isspecific to species and environment. The inclusionof M. phaseolina showed that the presence ofa pathogen triggered the release of additional and differ-ent compounds by IMC8 compared with the biocontrolagent alone. However, we cannot be certain that thepresence of E. pulchra would trigger the same com-pounds as M. phaseolina. In addition, the antimicrobialcompounds detected in this study have not been evalu-ated for pathogen suppression. A more thorough study isrequired on the role of volatile and non-volatile com-pounds from IMC8 in suppressing fungal pathogens.The SEM images show apparent parasitism of the

powdery mildew spores and hyphae by IMC8.Observations on untreated leaves show intact hyphaeand spores up to 5 days post-inoculation (Fig. 4a-e),indicating that the dehydration process for the SEMobservations did not interfere with the observations ofresults. Although the stepwise dehydration of leavesduring preparation for SEM was expected to somewhataffect the appearance of the spores and hyphae on leafsurfaces, the treatments did not impact the integrity ofthe powdery mildew fungus (Fig. 4). On leaves treatedwith the BCA at 48 h post-inoculation, the bacteria hadcolonized and lysed both the spores and hyphae of thepowdery mildew fungus (Fig. 5b-f). The BCA alsoseems to multiply on the leaf and on the fungal sporesand hyphae. These results support previous research

showing spore lysis caused by two other biocontrolagents (Serratia sp. and Stenotrophomonas sp.)(Mmbaga et al. 2016). The ability to colonize and multi-ply on the pathogen host would increase the bacterialinoculum level over and above that applied to the plant.This increased IMC8 population could then contribute tolonger-term control.The isolate IMC8 was identified as B. thuringiensis,

an organism well-known for its bioactivity against insectpests (Kumar et al. 1996; Roh et al. 2007). The Gram-positive B. thuringiensis is known as a soil-dwellingbacterium commonly used as an environmentallyfriendly biological pesticide, specific in its activity withlittle or no effect on humans, wildlife, pollinators, andmost other beneficial insects (Roh et al. 2007). Sporesand crystalline insecticidal proteins produced byB. thuringiensis have been used to control insect pestssince the 1920s and are often applied as liquid sprays(Kumar et al. 1996). Although the bacterium is known tooccur naturally on leaf surfaces and other environmentsincluding insect-rich environments, isolate IMC8 wasdetected on dogwood leaf surfaces and inside dogwoodstem tissue (Mmbaga et al. 2008; Lawrence 2012;Maheshwari et al. 2015). Its occurrence as an epiphyteas well as an endophyte of dogwood suggests that it mayhave a beneficial role on its dogwood host. The isolateIMC8 produced protein crystals that may have potentialbenefit in protecting dogwood plants against devastating

H

PM

PM

PM

PM

H

H

HPM

Fig. 4 Appearance of powdery mildew (Erysiphe pulchra) spores on untreated leaves (control) over a duration of (a) 3 h, (b) 6 h, (c) 15 h,(d) 48 h or (e) 5 d showing intact hyphae (H), and powdery mildew spore (PM) showing hyphae (H), and powdery mildew spore (PM).

E. Rotich et al. 8

dogwood borers, but this study did not evaluate its roleagainst these pests. B. thuringiensis produces a vastnumber of protein toxins, some of which have receivedlittle investigation and their broad-spectrum activity isnot known (Palma et al. 2014). This current studyshowed that foliar sprays of this organism have potentialapplication in the biological control of powdery mildew,one of the most important diseases of dogwood.

Acknowledgements

Authors would like to thank Drs. Richard Hall and RogerSauvé for their valuable contribution in manuscript reviews.

Funding

This work was supported by the USDA/NIFA [2010-38821-21477].

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