sign in sign up
<<
Home , Powdery mildew

Canadian Journal of

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

Biological control of powdery mildew on 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 on Cornus￿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: 08 Jul 2019. Published online: 31 Jul 2019.

Submit your article to this journal

Article views: 101

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tcjp20 Can. J. Plant Pathol., 2019 https://doi.org/10.1080/07060661.2019.1641555

Disease control/Moyens de lutte

Biological control of powdery mildew on Cornus florida using endophytic Bacillus thuringiensis

EMILY ROTICH1, MARGARET T. MMBAGA2 AND JACQUELINE JOSHUA3

1College of Sciences and Mathematics, Department of Biology, Belmont University, Nashville, TN 37212, USA 2College of Agriculture, Human and Natural Sciences, Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN 37209, USA 3Division of Crop Science, Monsanto Company, Chesterfield, MO, 63017, USA

(Accepted 4 July 2019)

Abstract: Powdery mildew ( pulchra) is a devastating disease in nursery production of flowering dogwood (Cornus florida L.). Disease management relies heavily on season-long use of chemical and there is a demand for alternative strategies. An endophytic bacterium IMC8 was isolated from disease-free C. florida stem tissue, and was shown to be an effective biological control agent against E. pulchra in growth chamber, greenhouse and shadehouse environments. This isolate, identified as Bacillus thuringiensis, exhibited normal growth in NaCl concentrations up to 6%, pH 5 to 11, and temperatures up to 50°C. It was compatible with the conventional thiophanate methyl and clarified hydrophobic extract of neem seed oil at label recommended concentrations. The isolate IMC8 produced volatile compounds identified by GC/MS, which included antifungal and antibacterial compounds and suggested antibiosis as a mode of action. Scanning electron microscopy revealed lysis of powdery mildew and hyphae, which suggested as a second mode of action. These observations suggested that the isolate IMC8 has potential as a biological control agent in the management of powdery mildew on C. florida, and would tolerate a wide range of salinity, pH and high temperature conditions. It could be included as a component of integrated management in rotation or tank mix with lower levels of conventional fungicides, or used with clarified hydrophobic extract of 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 (Cornus florida). La gestion des maladies repose largement sur l’utilisation de produits chimiques pendant toute la saison. Couramment, il existe une demande pour des stratégies alternatives au produit chimique. La recherche d’agents de lutte biologique contre l’oïdium a permis l’isolement d’une bactérie endophyte, IMC8, à partir de tissu de C. florida indemne de maladie. L’isolat IMC8 s’est avéré être un agent de lutte biologique efficace contre E. pulchra dans les environnements de chambre de culture, de serre et serre d’ombre. Cet isolat identifié comme Bacillus thuringiensis a présenté une croissance normale dans une large gamme de niveaux de salinité et de pH, allant jusqu’à 6% de NaCl et pH 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 le biofongicide, l’extrait hydrophobe clarifiéd’huile de graines de neem, à des concentrations couramment utilisées dans les applications de lutte contre l’oïdium au pepinere commercial. L’isolat IMC8 a produit des composés volatils identifiés par GC/MS, parmi lesquels des composés antifongiques et antibactériens ont suggéré l’antibiose comme mode d’action. D’ailleurs, la microscopie électronique à balayage a révélé la lyse des spores et des hyphes de l’oïdium, suggérant le parasitisme comme second mode d’action. Ces observations suggèrent que l’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 de conditions salines, de pH et de températures élevées. Les résultats présentés suggèrent que l’isolat IMC8 peut constituer une alternative aux fongicides 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: [email protected]

© 2019 The Canadian Phytopathological Society

Published online 31 Jul 2019 E. Rotich et al. 2 comme composante de la lutte intégrée en alternance, soit en mélange en réservoir de pulvérization avec des niveaux inférieurs de fongicides classiques, soit il peut être utilisé avec un extrait hydrophobe clarifiéd’huile de graines de neem.

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

few flowers for breeding purposes. In addition, break- Introduction down of resistance to powdery mildew or environmental Cornus florida (flowering dogwood) is a popular orna- effects which render the resistance ineffective at differ- mental tree throughout the southeastern United States. ent locations is a problem (Mmbaga and Sauvé 2004). Powdery mildew caused by Erysiphe (Sect. A few including ‘Cherokee Brave’, ‘Karen’s Microsphaera) pulchra (Cook & Peck, Braun & Appalachian Blush’, ‘Jean’s Appalachian Snow’, Takamatsu), is a devastating disease of C. florida ‘Kay’s Appalachian Mist’, and ‘Appalachian Joy’ have (Hagan and Mullen 1995; Mmbaga 1998, 2000;Li some degree of resistance to powdery mildew, but none et al. 2009). The white, powdery appearance caused by has a high level of resistance. An integrated disease dust-like mycelium on leaf surfaces reduces the aesthetic management system that combines different methods value of infected trees, as well as the rate of photosynth- would be ideal to manage powdery mildew infections esis, causing stunted growth and increasing the time (Witte et al. 2000; Li et al. 2009). needed for plants to reach optimal size for plant sale The use of biological and biorational products to sup- (Windham 1996; Mmbaga and Sauvé 2004; Agrios press fungal pathogens has been recommended in horticul- 2005; Shi et al. 2008). Severe infection with powdery tural crop production due to their lower level of toxicity mildew has also been shown to reduce flower set and the (Stimmel 1996). There are a few biorational products that amount of fruit formed (Heald 1999). are labeled for powdery mildew control and include plant Since the emergence of powdery mildew on flowering extracts from neem oil marketed as Triact 70, and neem dogwood in the early 1990s, management strategies have Gold as well as potassium bicarbonate salts marketed as relied heavily on conventional fungicides with applica- Armicarb and Kaligreen (Mmbaga and Sheng 2002). The tions starting soon after bud-break and continuing all incorporation of Armicarb and Triact 70 in fungicide rota- season-long (Windham 1994; Mmbaga 2000). tions has been reported to reduce conventional fungicide Fungicide applications such as wettable sulfur primarily usage by up to 56% (Mmbaga and Sheng 2002). Biological are used preventatively, but they are not effective against control strategies which use one living organism to control existing infections (Ash 1999). However, other fungi- the growth and proliferation of another are safer and inex- cides including thiophanate methyl and propiconazole pensive alternatives to conventional agrochemicals (Van are commonly used all season long as protective as Driesche and Bellow 1996; Hawley and Eitzen 2001; well as curative remedies. The season-long use of che- Gardener and Fravel 2002; Kiss 2004; Sharma et al. mical fungicides has significantly increased the cost of 2011). Biological-based disease management can be inte- production, leading to abandonment of C. florida pro- grated with other strategies such as host resistance and low duction by small-scale growers in favor of other orna- levels of fungicides. Since bio-control agents are living mentals (Li et al. 2009). In addition, there are concerns organisms, it is important to investigate the conditions over accidental exposure of humans and wildlife to these necessary for their optimal development, survival and effi- chemicals, environmental contamination, adverse effects cacy (Bélanger and Labbé 2002). Screening of diverse on microbial biodiversity, and beneficial non-target nat- epiphytes for biological control agents for dogwood pow- ural microflora that otherwise protect plants against other dery mildew identified bacteria, fungi and yeasts that sup- pathogens (de Jager et al. 1995; Sharma et al. 2011). pressed powdery mildew (Mmbaga et al. 2008, 2016; Continued use of fungicides also contributes to the Mmbaga and Sauvé 2009). Screening of diverse endo- potential for development of resistance in pathogens phytes identi fied the bacterial isolate IMC8 to be effective (Sharma et al. 2011). There is a demand for safer, non- in suppressing powdery mildew (E. pulchra) in flowering chemical alternatives for powdery mildew management. dogwoods. This study focused on the evaluation of the Although genetic disease resistance is an environmen- efficacy of isolate IMC8 in limiting powdery mildew dis- tally safe and affordable approach for disease manage- ease severity, its identification and characterization. The ment, it takes a long time to develop resistant genotypes, study also evaluated this isolate in different environmental especially on tree crops. Flowering dogwood seedlings conditions and assessed its potential mode of action in require four years to initiate flowering and then produce powdery mildew control. Biological control of powdery mildew on Cornus florida 3

Materials and methods Dayton, NJ) and water were used as the positive and negative controls, respectively. Treatments with isolate Evaluation of the impact of isolate IMC8 on powdery IMC8 were initiated as soon as the initial symptoms of mildew disease severity powdery mildew were observed in the local area in early The effect of IMC8 on the severity of powdery mildew to mid-May. The experiments were arranged in on flowering dogwood was evaluated using (i) a randomized complete block design with a replication a detached leaf technique in a moist chamber set at of 40 individual plants per treatment in greenhouse 23 ± 3°C and 99–100% relative humidity, (ii) 3-month- experiments and eight individual plants per treatment in old C. florida seedlings grown in a greenhouse environ- shadehouse experiments. Previously infected plants ran- ment at 26–28°C temperature in McMinnville (Nursery domly placed in greenhouse and shadehouse experimen- Research Center) and in Nashville’s University Research tal areas provided a continuous source of air-borne Facility, and (iii) two year old seedlings of three geno- powdery mildew inoculum. Plants were sprayed with types ‘Cherokee Princess’ (susceptible), ‘R14ʹ and ‘MI9ʹ treatments to run-off every 7–10 days, and treatment (moderately resistant) grown in a shadehouse environ- applications were terminated at the end of August, coin- ment under 50% shadecloth. For the detached leaf tech- ciding with the timing of grower practices in powdery nique, leaves of similar size were harvested from disease mildew spray programs. Disease severity was assessed free C. florida “Cherokee Princess” and surface- as described above. Data were analyzed using Statistical sterilized with 10% alcohol for 60s, rinsed twice in Analysis System (SAS) software v. 9.4 (SAS institute sterilized water, and blotted dry using heat sterilized Inc., Cary, NC), with t-tests to compare means following tissue paper. The leaves were then placed in a moist PROC ANOVA and PROC GLM. Least significant dif- chamber lined with a double layer of sterilized tissue ferences (LSD) were calculated at P ≤ 0.05 (Ott and paper kept moist using sterilized water. The endophytic Longnecker 2001). bacterium IMC8 previously isolated from flowering dog- wood stems, and maintained in nutrient agar (NA) or Luria-Bertani (LB) agar at −80°C was retrieved as an Identification and characterization of isolate IMC8 inoculum source. The inoculum suspension of approxi- mately 106 CFU/mL was prepared from 36-h-old cul- The bacterial isolate was identified using molecular tures by the plating technique and inoculum sequence analysis, as well as morphological, and phy- concentrations were assessed using previously standar- siological characteristics based on colony shape and size dized odometer readings corresponding to 106 CFU/mL. (Janda and Abbott 2002). Bacterial DNA was extracted Detached leaves were spray inoculated using sterilized from the isolate using a DNA blood and tissue kit hand-held atomizers. Non-treated controls were sprayed (Qiagen Inc, Valencia, CA) following the manufacturer’s with sterilized water. A replication of four leaves per recommendations (DNeasy Blood and tissue kit treatment arranged in a randomized complete block Handbook, July 2006). The DNA concentration and design was used. At two hours after inoculation with relative purity was determined using a Nanodrop Lite IMC8, the detached leaves were brush inoculated with (Thermo Fisher Scientific, Wilmington, DE). A pair of powdery mildew spores from previously infected leaves oligonucleotide primers (RW01: 5ʹ-AAC TGG AGG and incubated at 23 ± 3°C. Disease severity was assessed AAG GTG GGG AT-3ʹand DG74: 5ʹ-AGG AGG TGA 14 d after inoculation using a modified Horsfall and TCC AAC CGC A-3ʹ) universal for bacteria (Greisen Barratt (1945) rating scale of 0 to 5, where 0 = no et al. 1994) were used to amplify a 370 bp region of the disease symptoms; 1 = 1–10%; 2 = 11–25%; 16S rRNA gene. PCR amplification was carried out in 3=26–50%; 4 = 51–75%; and 5 = 76–100% of leaves a final volume of 25 μL with PCR buffer 1X (Promega, with disease symptoms. The experiment was performed Madison, WI), 2.5 mM MgCl2, 200 μM of each dNTP, twice and the two years’ data were pooled. 100 pM of each primer, 20 ng of genomic DNA and 2.5 The effect of IMC8 on powdery mildew was also units of Taq polymerase (Promega, Madison, WI). evaluated in greenhouse experiments using 3-month-old Amplification was carried out in a PTC 100 Thermal C. florida seedlings and in shadehouse experiments Cycler® (Boier Lifepro, Grand Island, NY) programmed under 50% shadecloth using 2-year-old grafted plants with an initial denaturation temperature of 95°C for of three genotypes ‘Cherokee Princess’, ‘R14ʹ and 5 min, followed by 34 cycles consisting of denaturation ‘MI9ʹ grown in 3.78 L containers. The fungicide thio- at 95°C for 1 min, annealing of the primers at 55°C for phanate methyl (Cleary’s 3336F, Cleary Chemicals, 1 min, and a final extension step at 72°C for 10 min. E. Rotich et al. 4

The PCR product was purified using an Exosap DNA thiophanate methyl (Cleary 3336 F) and biorational pro- purification kit (Invitrogen, Life Technologies, Inc. ducts, potassium bicarbonate (Armicarb100) and clari- Carlsbad, CA) following the manufacturer’s recom- fied hydrophobic extract of neem oil (Triact 70) Dhingra mended protocols. The PCR products sequenced at and Sinclair 1995) were previously tested using slight Davis Sequencing Inc. (Davis, CA). A BLAST search variations of label recommended rates. The most effica- (Altschul et al. 1997) was performed with GenBank cious rates that did not harm dogwood plants were (ncbi.nlm.nih.gov/genbank/) sequence data libraries, selected for this study (Mmbaga and Sheng 2002). and the closest similarity match with other 16S rDNA Application rates of thiophanate methyl were 0.78 mL/ gene sequences previously deposited in GenBank was L, and potassium bicarbonate and neem oil extract were used to determine the identity of the bacterium. The at 6.6 g/L. These fungicides were dissolved in sterilized DNA sequence was deposited in GenBank (accession milli Q water and mixed into the NA just before pouring no. MK245974). the agar into the Petri plates. The CFU/mL was assessed up to a 72 h growth period.

Effect of different environmental conditions on the bacterial growth Potential mode of action of IMC8 The isolate was grown on nutrient broth (Difco Becton Dickinson, Sparks, MD), adjusted to 106 CFU/mL and This study evaluated the possible involvement of (i) then serially diluted six-fold before plating to determine volatile compounds produced by the endophyte and (ii) its growth at different levels of salinity and pH, and in parasitism of the powdery mildew by IMC8 media amended with fungicides that are commonly used isolate. in powdery mildew control. At the end of each study, Gas chromatography mass spectrometry (GC/MS) was colonies were counted to determine the number of col- used to detect volatile compounds from IMC8. For this ony forming units (CFU) at each condition. These analysis, a single colony of the isolate was grown on experiments used four replicates and each study was nutrient agar for 48 h in glass vials covered with a poly- performed twice; the data were pooled for analysis. propylene hole cap top (Supelco, Bellefonte, PA) to Log10 numbers of CFU/mL were analyzed using SAS. maximize the trapping of volatiles. The vials pre- To evaluate the effect of temperature, the bacterial concentrated the volatiles and allowed identification isolate was streaked onto nutrient agar (NA) Petri plates, and quantification of even small amounts, which were and incubated at 4, 23, 28, 37, 45 and 50°C to determine detected through the solid phase microextraction growth and morphological characteristics at the 24 h-old (SPME) method and analyzed by GC/MS. The analysis culture stage. Growth was monitored for 72 h, and was performed using a Varian 3800 gas chromatograph scored as: – no growth, + slight growth with up to 102 equipped with an ion-trap mass spectrometer Varian CFU/mL, ++ moderate growth (103 to 106 CFU/mL), Saturn 2000 (Agilent technologies, Walnut Creek CA). and +++ good growth (with more than 106 CFU/mL). The solid phase micro-extraction (SPME) fiber 50/30 μm To determine the survival of the bacterial isolate in DVB/Carboxen/PDMS StableFlex for manual holder different concentrations of sodium chloride (NaCl), the (Supelco, Bellefonte, PA) was conditioned by running bacterium was grown in nutrient broth for 24 h and it through the programmed cycle before exposing it to streaked on NA plates containing 2, 4, 6, 8 and 10% the samples. The fiber was exposed into the headspace of w/v NaCl with 6 plates per salinity level. All plates were the vial for 10 min, retracted, and then introduced into incubated at room temperature for 48 h and bacterial a GC injector where the trapped volatiles were desorbed growth was quantified using the previously developed from the fiber to a chromatography column, using growth curve of CFU/mL in serially diluted plates. The Helium as a carrier. The injector temperature was set at bacterial isolate was also plated on NA with the pH 220°C, and detector temperature at 265°C. Desorption in adjusted to 5, 7, 9, 11, and 12 using hydrochloric acid the hot injector was programmed with an initial tempera- and sodium hydroxide. The number of colonies was ture of 28°C held for 1 min, and then gradually increased quantified and expressed as CFU/mL. to 180°C, with a total run time of 40 min. The volatile Fungicide tolerance was evaluated to determine com- secondary metabolites released were identified using the patibility of IMC8 with conventional fungicides and two chromatograph’s library search by comparing with the biorational products labeled for integrated management database in the National Institute of Standards and of powdery mildew in C. florida. The fungicide Technology (www.nist.gov). Biological control of powdery mildew on Cornus florida 5

While previous reports provided evidence that endo- gold-palladium (Cressington sputter coater, Ted Pella phytes synthesize many secondary metabolites, some Inc, Redding, CA) for 90 seconds. Following conductive metabolites produced may be different in the presence silver coating (Electron Microscopy Sciences, Hatfield, of a pathogen (Tan and Zou 2001). However, as an PA), the samples were subjected to SEM (Quanta 250 obligate parasite, E. pulchra cannot be grown on artifi- Environmental Scanning Electron Microscope, Hitachi cial medium. Thus, to determine if isolate IMC8 pro- S4200, Hillsboro, Oregon) to view the interaction of duced different volatiles in the presence of a pathogen, IMC8 with the fungal hyphae and spores. this study used a facultative root rot pathogen with a broad host range, Macrophomina phaseolina. This pathogen, the causal agent of charcoal rot disease, affect Results and discussion a diversity of crops including dogwood (Smith and Bega 1964; Seymour 1969; Mmbaga et al. 2018) and IMC8 Powdery mildew development is highly influenced by has displayed efficacy against this pathogen in a different weather conditions. The growth chamber environment study (Joshua and Mmbaga, unpublished). The study had a moderate temperature and high relative humidity comprised three treatments with three vials containing known to favor the disease (Agrios 2005). The isolate (i) a 5 mm plug of IMC8 alone from a 24 h-old culture, IMC8 was applied before powdery mildew was intro- (ii) a 5 mm mycelial plug of M. phaseolina from 5 day- duced and it was highly effective in controlling the old cultures and a 5 mm plug of IMC8 endophyte and disease, with only traces of powdery mildew observed pathogen, and (iii) a 5 mm plug of M. phaseolina alone. on the leaf surface. In the water control treatment, how- The tests were done in duplicate and repeated once. All ever, powdery mildew covered 100% of the leaf sur- samples were incubated at 28°C for 48 h prior to con- faces. This observation suggests that the moist chamber ducting the sample analysis using GC/MS. environment is highly favorable to IMC8 proliferation as The possible parasitism of the powdery mildew fun- well as to the powdery mildew pathogen. gus by IMC8 was examined using the detached leaf Results from the greenhouse experiments are pre- technique in moist chambers in which powdery mildew sented in Figs 1-2. The first experiment in Nashville infected leaves of similar size, age and physical appear- had a higher disease severity and IMC8 was not effective ance were collected from a C. florida tree. The leaves (Fig. 1). In the second experiment in Nashville, powdery were placed in a moist chamber lined with a double layer mildew severity was high, IMC8 reduced powdery mil- of sterilized tissue paper kept moist using sterilized dew but its effect was not statistically different from water. The detached leaves were spray inoculated with water control (Fig. 1). All three of the host genotypes an IMC8 inoculum suspension of approximately 106 evaluated developed moderate disease severity with no CFU/mL prepared from 36 h-old cultures using sterilized statistical differences between them. However, there hand-held atomizers. Non-treated controls were sprayed were significant differences between treatments in all with sterilized water. Five treatments represented time- three genotypes. Treatment with the fungicide was points when leaves were harvested after inoculation with most effective in controlling powdery mildew followed IMC8 and then fixed using Formalin–acetic acid–alcohol by IMC8 as a biological control agent; the highest pow- (FAA) overnight and stored at 4°C until processing for dery mildew severity was observed on the water control scanning electron microscopy (SEM). The time-points (Fig. 2). The isolate IMC8 provided significant control of included 3 h, 6 h, 15 h, 48 h and 5 days post- powdery mildew compared with the water controls, but inoculation and each time-point was replicated with it was less effective than the conventional fungicide. four leaves; the study was repeated once. These results suggest that IMC8 may be used as part of an integrated disease management plan that could include moderately resistant cultivars or low levels of compatible fungicides. Leaf dehydration process for SEM Based on the molecular tests, isolate IMC8 shared The samples were dehydrated in 30% ethanol for 15 min, 99% similarity with Bacillus sp. (B. thuringiensis 50% ethanol for 15 min, 75% ethanol for 15 min, 85% (NR_102506.1), B. cereus (NR_074540.1) and ethanol for 15 min, 95% ethanol for 15 min, followed by B. anthracis (NR_074453.1). The three species have pure ethanol three times for 15 min. Following the step- the same cell size and morphology and form oval spores. wise dehydration, samples were critical point dried in Further tests were conducted to differentiate the three CO2 (Samdri®-PVT-3D, Tousimis, Rockville, MD), Bacillus species biochemically: (i) the motility of the mounted onto sample stubs, and sputter coated with bacterial cells was visualized microscopically at 1000x E. Rotich et al. 6

5 aa Fungicide IMC8 4 Water Control a a

3 b

2 b

1 b b c 0 Disease severity (0-5 scale) Disease severity McMinnville Nashville Nashville 1tpxE 2tpxE

Fig. 1 (Colour online) Powdery mildew severity on Cornus florida seedlings after foliar application of the bacterial biocontrol agent IMC8 as compared with the fungicide thiophanate methyl and a water control under greenhouse conditions at two locations (McMinnville and Nashville). Similar results were obtained in McMinnville Experiment 2 (not shown). Means within a group followed by the same letter are not significantly different at p ≥ 0.05.

5 B. thuringiensis from B. cereus, the IMC8 bacteria were Fungicide control IMC8 Water control a stained for the detection of parasporal crystals associated 4 aawith B. thuringiensis (Ejiofor and Johnson 2002). The b test showed that IMC8 produces intracellular parasporal crystals in association with formation. This test 3 b b eliminated B. cereus and confirmed that isolate IMC8 2 is B. thuringiensis (Fig. 3). The isolate formed endo- spores, an important feature for the microbial survival in stressful environments (Reva et al. 2004), and which 1 Disease severity (0-5 scale) Disease facilitates long-term storage and product formulation. c c c Abundant colonies of the bacterium developed at 0 CP Genotypes MI R14 a wide range of temperatures from 23°C to 50°C with an optimum temperature of 28°C. These observations Fig. 2 (Colour online) Powdery mildew severity on three Cornus suggest that IMC8 has potential application in agricul- florida genotypes, including the susceptible ‘Cherokee tural production at a wide range of temperatures. Growth Princess’ (CP) and two moderately resistant selections MI9 and of IMC8 was reduced significantly starting at 4% NaCl ° R14, grown in a greenhouse at 26–28 C in Nashville, TN, and treated w/v and was further reduced at 6% compared with the with foliar sprays of the bacterial isolate IMC8, the fungicide thio- control; its growth was completely inhibited above 8% phanate methyl or water (control) every 14 d over a 3 month growing season. Treatment means within a group followed by the same letters NaCl w/v. High soil salinity is an abiotic stress that often are not significantly different at p ≥ 0.05. limits crop production in affected areas. Bacillus species have been reported to be growth promoting (data not presented) and also have been found to enhance plant using cultures grown in nutrient broth maintained at 30° tolerance to abiotic stresses, leading to improved plant C in a shaker set at 125 rpm for 3 h. Results were growth and production (Yang et al. 2009). Arid regions positive for motility; (ii) growth on nutrient agar supple- are especially impacted by high soil salinity and biolo- mented with 5% sheep red blood cells showed gical control agents may be useful in increasing plant a hemolysis pattern. (iii) An additional test for growth tolerance to high soil salinity. Soil salinity levels affect- in 0.5 IU penicillin revealed positive results for penicil- ing plant growth have an electrical conductivity (EC) of lin resistance and negative for the ‘String of Pearls’ > 4 dS/m which corresponds to approximately 2.3% reaction, which is the impairment of the cell wall that NaCl (U.S. Salinity Laboratory Staff 1954). In this Bacillus anthracis exhibits in the presence of as little as study, the bacterial isolate was able to grow at 4% 0.05 IU penicillin (Chamberlain 2015). The three tests NaCl and up to 6% NaCl, indicating an ability to survive eliminated the possibility of B. anthracis. To distinguish in high saline conditions. There is a need to further Biological control of powdery mildew on Cornus florida 7

Fig. 3 (Colour online) Presence of crystal proteins in bacterial cells of isolates IMC8 treated with a differential crystal stain. (A) Light microscopy image of a buffalo black-stained spore-crystal mixture consistent with Bacillus thuringiensis. Vegetative cells are stained (red arrow), black structures are crystal proteins (black arrow). explore its potential supplementary role in improving isolate IMC8, and also from a combination of IMC8 and plant tolerance to high salinity. the pathogen M. phaseolina (Table 1). The volatile com- The isolate was able to grow at different pH levels pounds produced by IMC8 included cyclopentasiloxane ranging from 5 to11, with a significant reduction in decamethyl, an active antimicrobial compound (Baishya growth at pH 12. A neutral pH favors growth of most et al. 2018). However, most of the antifungal compounds organisms and increased pH may impact plant growth were produced in the presence of a pathogen (Table 1), (Margesin and Schinner 2001). The current results show including cycloheptasiloxane tetradecamethyl, cyclooc- a potential role of the bacterial isolate in helping to offset tasiloxane hexadecamethyl, and heptadecane, 2,6,10,14- the impact of low or high pH on plant growth, but tetramethyl, which are known antimicrobials (Barakat additonal studies are needed. Previous studies reported 2011; Keskin et al. 2012; Moustafa et al. 2013; Jasim by Yang et al. (2009) showed that some bacteria help et al. 2015). Mackie and Wheatley (1999) reported that plants tolerate abiotic stress. The results show that the growth of isolate IMC8 fi increased signi cantly in the presence of thiophanate Table 1. Volatile compounds emitted by the IMC8 isolate alone or methyl (p < 0.05) compared with the control. This in the presence of Macrophomina phaseolina (MP) as identified strongly suggests that the isolate and thiophanate methyl by GC/MS SPME. can be used together, or in rotation as part of an integrated Sourcea Compound Functionb powdery mildew management strategy. To our knowl- edge, this is the first report on such BCA/fungicide com- Growth medium Control N/A patibility. However, growth of the isolate was suppressed Fungicide Methoxy-phenyl- Antifungal oxime completely by the presence of potassium bicarbonate, but (thiophanate not by neem oil extracts, both of which are commercial methyl) biorational products. These results suggest that isolate IMC8 Cyclopentasiloxane Antimicrobial compound IMC8 can be used in combination with thiophanate decamethyl (Keskin et al. 2012; Baishya fi et al. 2018) methyl or clari ed hydrophobic extract of neem oil to IMC8 + MP Cycloheptasiloxane Antimicrobial (Barakat 2011; reduce fungicide use in dogwood nursery production, tetradecamethyl Moustafa et al. 2013) but not in combination or in rotation with potassium Cyclooctasiloxane, Antimicrobial (Jasim et al. bicarbonate. Mmbaga and Sheng (2002) reported 56% hexadecamethyl 2015) Heptadecane, Antimicrobial (Manilal and potential reduction in conventional fungicide usage by 2,6,10,14- Idhayadhulla 2014) incorporating Armicarb or Triact70 in fungicide rotations tetramethyl as part of integrated disease management. a fi The GC-MS spectra showed various chromatographs Refers to the source of the compound identi ed. bRefers to the putative function of the compound. signifying different secondary metabolites produced by E. Rotich et al. 8 the response of fungi to bacterial volatile compounds is showing spore lysis caused by two other biocontrol specific to species and environment. The inclusion agents (Serratia sp. and Stenotrophomonas sp.) of M. phaseolina showed that the presence of (Mmbaga et al. 2016). The ability to colonize and multi- a pathogen triggered the release of additional and differ- ply on the pathogen host would increase the bacterial ent compounds by IMC8 compared with the biocontrol inoculum level over and above that applied to the plant. agent alone. However, we cannot be certain that the This increased IMC8 population could then contribute to presence of E. pulchra would trigger the same com- longer-term control. pounds as M. phaseolina. In addition, the antimicrobial The isolate IMC8 was identified as B. thuringiensis, compounds detected in this study have not been evalu- an organism well-known for its bioactivity against insect ated for pathogen suppression. A more thorough study is pests (Kumar et al. 1996; Roh et al. 2007). The Gram- required on the role of volatile and non-volatile com- positive B. thuringiensis is known as a soil-dwelling pounds from IMC8 in suppressing fungal pathogens. bacterium commonly used as an environmentally The SEM images show apparent parasitism of the friendly biological pesticide, specific in its activity with powdery mildew spores and hyphae by IMC8. little or no effect on humans, wildlife, pollinators, and Observations on untreated leaves show intact hyphae most other beneficial insects (Roh et al. 2007). Spores and spores up to 5 days post-inoculation (Fig. 4a-e), and crystalline insecticidal proteins produced by indicating that the dehydration process for the SEM B. thuringiensis have been used to control insect pests observations did not interfere with the observations of since the 1920s and are often applied as liquid sprays results. Although the stepwise dehydration of leaves (Kumar et al. 1996). Although the bacterium is known to during preparation for SEM was expected to somewhat occur naturally on leaf surfaces and other environments affect the appearance of the spores and hyphae on leaf including insect-rich environments, isolate IMC8 was surfaces, the treatments did not impact the integrity of detected on dogwood leaf surfaces and inside dogwood the powdery mildew fungus (Fig. 4). On leaves treated stem tissue (Mmbaga et al. 2008; Lawrence 2012; with the BCA at 48 h post-inoculation, the bacteria had Maheshwari et al. 2015). Its occurrence as an epiphyte colonized and lysed both the spores and hyphae of the as well as an endophyte of dogwood suggests that it may powdery mildew fungus (Fig. 5b-f). The BCA also have a bene ficial role on its dogwood host. The isolate seems to multiply on the leaf and on the fungal spores IMC8 produced protein crystals that may have potential and hyphae. These results support previous research benefit in protecting dogwood plants against devastating

H H PM PM PM H

PM

H PM

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). Biological control of powdery mildew on Cornus florida 9

H PM BCA

PM BCA BCA

H BCA H PM BCA

Fig. 5 Appearance of powdery mildew (Erysiphe pulchra) spores and hyphae at (a) 3 h, (b) 6 h, (c) 15 h, (d) 48 h or (e) 5 d post-treatment with IMC8 as a biological control agent (BCA), showing lysed hyphae (H) in (d and e), and spores (PM) colonized by IMC8 in (d, e). dogwood borers, but this study did not evaluate its role Baishya MK, Saika KK, Hazarika NK, Baishya D, Das DJ. 2018. against these pests. B. thuringiensis produces a vast Antimicrobial potential on in vitro cytotoxicity study of Tabernaemintana divaricata (L.) stem bark extract against HEK 293 number of protein toxins, some of which have received cell line. J Pharmacy. 8:11–18. little investigation and their broad-spectrum activity is Barakat DA. 2011. Insecticidal and antifeedant activities and chemical not known (Palma et al. 2014). This current study composition of Casimiroa edulis La Llave & Lex (Rutaceae) leaf extract showed that foliar sprays of this organism have potential and its fractions against Spodoptera littoralis larvae. Aust J Basic Appl Sci. 5:693–703. application in the biological control of powdery mildew, Bélanger RR, Labbé C. 2002. Control of powdery mildews without one of the most important diseases of dogwood. chemicals: prophylactic and biological alternatives for horticultural crops. In: Bélanger RR, Bushnell WR, Dik AJ, Carver TLW, edi- tors. The powdery mildews: a comprehensive treatise. St. Paul (MN): Acknowledgements APS Press; p. 256–267. Chamberlain N. 2015. Bacillus anthracis: string of pearls reaction. Authors would like to thank Drs. Richard Hall and Roger [accessed 2018 Feb 20]. www.asmscience.org/content/education/image gallery/image.4018. Sauvé for their valuable contribution in manuscript reviews. de Jager CM, Butot RPT, Klinkhamer PGL, de Jong TJ, Wolff K, van der Meijden E. 1995. Genetic variation in chrysanthemum for resistance to Frankliniella occidentalis. Entomol Exp Appl. Funding 77:277–287. Dhingra OD, Sinclair JB. 1995. Basic plant pathology methods. Boca This work was supported by the USDA/NIFA [2010- Raton (FL): CRC Press, Inc; p. 132–163. 38821-21477]. Ejiofor AO, Johnson T. 2002. Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States. J Indust Microbiol Biotech. 28:284–290. References Gardener BBM, Fravel DR. 2002. Biological control of plant pathogens: research, commercialization, and application in the USA. Plant Health Agrios GN. 2005. Plant pathology. 5th ed. San Diego (CA): Elsevier Prog. 3:17. Online. Academic Press. Greisen K, Loeffelholz M, Purohit A, Leong D. 1994. PCR primers Altschul SF, Madden TL, Schäffer AA, Zhang JI, Zhang Z, Miller W, and probes for the 16S rRNA gene of most species of pathogenic Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of bacteria, including bacteria found in cerebrospinal fluid. J Clin protein database search programs. Nucleic Acids Res. 25:3389–3402. Microbiol. 32:335–351. Ash C. 1999. Powdery mildew on ornamental plants. [accessed 2018 Feb Hagan A, Mullen J. 1995. Controlling powdery mildew on ornamentals. 20]. http://www.extension.umn.edu/garden/yard-garden/flowers/pow Alabama Coop. Ext. Sys. Cir. ANR-407. dery-mildew-on-ornamental-plants/. E. Rotich et al. 10

Hawley RJ, Eitzen JEM. 2001. Biological weapons- a primer for Mmbaga MT, Sauvé RJ, Mrema FA. 2008. Identification of microor- microbiologists. Ann Rev Microbiol. 55:235–253. ganisms for biological control of powdery mildew in Cornus florida. Heald AD. 1999. [accessed 2019 Apr 2]. https://news.ca.uky.edu/article/ Biol Control. 44:67–72. powdery%20mildew%20threatens%20dogwoods.%20SAS%20institute Mmbaga MT, Sheng H. 2002. Evaluation of biorational products for Horsfall JG, Barratt RW. 1945. An improved grading system for mea- powdery mildew management in Cornus florida. J Environ Hortic. suring plant diseases. Phytopathology. 35:655. (Abstr.). 20:113–117. Janda JM, Abbott S. 2002. Bacterial identification for publication: when Moustafa MFM, Alamri SA, Taha TH, Alrumman SA. 2013. In vitro is enough enough? J Clin Microbiol. 40:1887–1891. antifungal activity of Argemone ochroleuca sweet latex against some Jasim H, Hussein AO, Hameed IH, Kareem MA. 2015. pathogenic fungi. Afr J Biotech. 12:1132–1137. Characterization of alkaloid constitution and evaluation of antimicrobial Ott RL, Longnecker M. 2001. An introduction to statistical methods and activity of Solanum nigrum using gas chromatography mass spectro- data analysis. Pacific Grove (CA): Dexbury Press. metry (GC-MS). J Pharmacognosy Phytother. 7:56–72. Palma L, Muñoz D, Berry C, Murillo J, Caballero P. 2014. Bacillus Keskin D, Ceyhan N, Uğur A, AD D. 2012. Antimicrobial activity and thuringiensis toxins: an overview of their biocidal activity. Toxins. 6:3296– chemical constitutions of West Anatolian olive (Olea europaea L.) 3325. leaves. J Food Agri Environ. 10:99–102. Reva ON, Dixelius C, Meijer J, Priest F. 2004. Taxonomic character- Kiss L. 2004. How dangerous is the use of fungal biocontrol agents to ization and plant colonizing abilities of some bacteria related to Bacillus non-target organisms? New Phytol. 163:453–455. amyloliquefaciens and Bacillus subtilis. FEMS Microbiol Ecol. Kumar PA, Sharma RP, Malik VS. 1996. The insecticidal proteins of 48:249–259. Bacillus thuringiensis. Adv Appl Microbiol. 42:1–43. Roh JY, Choi JY, Li MS, Jin BR, Je YH. 2007. Bacillus thuringiensis Lawrence S. 2012. Endophytic colonization of flowering dogwood as a specific, safe, and effective tool for insect pest control. J Microbiol (Cornus florida L.) and their potential applications [thesis]. Nashville Biotechnol. 17:547–559. (TN): Tennessee State University. Seymour CP 1969. Charcoal root disease. In: Peterson GW, Smith, Jr Li Y, Mmbaga MT, Windham AS, Windham MT, Trigiano RN. 2009. RS. Tech. Coordinators. Forest Nursery Diseases in the United States; p. Powdery mildew of dogwoods: current status and future prospects. Plant 11–13. U.S. Dep. Agric, Forest Service Agric Hndbk. 470. Dis. 93:1084–1092. Sharma N, Sharma KP, Gaur RK, Gupta VK. 2011. Role of Chitinase Mackie AE, Wheatley RE. 1999. Effects and incidence of volatile in plant defense. Asian J Biochem. 6:29–37. organic compound interactions between soil bacterial and fungal Shi A, Kantartzi S, Mmbaga MT, Chen P. 2008. Differentiation of two isolates. Soil Biol Biochem. 31:375–385. pathogens of powdery mildew disease in flowering dogwood (Cornus Maheshwari A, Mmbaga MT, Joshua J, Gurung S, Alyamani A. florida) by PCR-mediated method based on ITS sequences. 2015. Diversity of endophytes in flowering dogwoods and their potential J Phytopathol. 157:274–279. applications. Proc SNA Res Conf. 60:158–164. Smith RS, Bega RV. 1964. Macrophomina phaseolina in the forest tree Manilal A, Idhayadhulla A. 2014. Potential in vitro antimicrobial effi- nurseries of California. Plant Dis Rep. 48:206. cacy of Holigarna arnottiana (Hook F). Asian Pac J Trop Biomed. Stimmel JF. 1996. Biorational controls: problem-free? Regul Hortic. 22:13. 4:25–29. TanRX,ZouWX.2001. Endophytes: a rich source of functional Margesin R, Schinner F. 2001. Biodegradation and bioremediation of metabolites. Nat Prod Rep. 18:448–459. hydrocarbons in extreme environments. Appl Microbiol Biotechnol. U.S. Salinity Laboratory Staff. 1954. Diagnosis and improvement of 56:650–663. saline and alkali soils. Washington (DC): U.S. Government Printing Mmbaga MT. 1998. Effect of powdery mildew on growth of dogwood. Office. USDA Agricultural Handbook No. 60. Tenn. Nurserymen’s Assoc Newsl. 8:16–17. Van Driesche RG, Bellow TS, editors. 1996. Biological control agents for Mmbaga MT. 2000. Winter survival and source of inoculum of powdery plant pathogens. In: Biological control. Boston (MA): Springer; p. 93–101. mildew of dogwood. Plant Dis. 84:574–579. Windham AS. 1994. Disease management of woody ornamentals in Mmbaga MT, Mackasmiel LM, Mrema FA. 2018. Flowering dogwood nurseries and commercial landscapes. Univ. of Tennessee Agr. infections with Macrophomina phaseolina. Hortic Sci. 53:334–336. Extension Publication 1234. Mmbaga MT, Mrema FA, Mackasmiel LM, Rotich E. 2016. Effect of Windham MT. 1996. Resistance to powdery mildew in flowering bacteria isolates in powdery mildew control in flowering dogwoods dogwood. Proc SNA Res Conf. 41:195–199. (Cornus florida L.). Crop Prot. 89:51–57. Witte WT, Windam MT, Windham AS, Hale FA, Fare DC, Mmbaga MT, Sauvé RJ. 2004. Multiple disease resistance in dogwoods Clatterbuck WK. 2000. Dogwoods for American gardens. (Cornus sp.) to foliar pathogens. J Arboric. 30:101–107. University of Tennessee Agr Extension service. PB1670-30M-12/00 Mmbaga MT, Sauvé RJ. 2009. Epiphytic microbial communities on R12-4910-19-001-01. foliage of fungicide treated and non-treated flowering dogwoods. Biol Yang J, Kloepper JW, Ryu C. 2009. Rhizosphere bacteria help plants Control. 46:97-104. tolerate abiotic stress. Trends Plant Sci. 14:1–4.