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1 EVALUATION OF SILICON AND BIOFU NGICIDE PRODUCTS FOR MANAGING POWDERY MILDEW CAUSED BY Podosphaera fusca IN GERBERA DAISY ( Gerbera jamesonii ) By CATALINA MOYER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007
2 2007 Catalina Moyer
3 To my husband Scott
4 ACKNOWLEDGMENTS Immense appreciation given to Dr. Natalia Peres for her trusts and support. Thanks go out to the staff and faculty at the Gulf Coast Research and Education Center for their assistance. Especially thanks offered to Dr. Steve Mackenzie for his st atistical wisdom, for cari ng about my project and for inspire me as a scientist. Thanks left to my friends in Gainesville in particular Linley for sharing her home and Norma for sharing the goo d and stressful times as graduate students. Thanks go out to all other members of my commi ttee for their contributions to this project. Bountiful gratitude presented to my husband, for supporting me during the last two years. Thanks sent to my family for inspiring my growth.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 2 LITERATURE REVIEW.......................................................................................................13 Powdery Mildew................................................................................................................. ....13 Taxonomy and Phylogenetic History..............................................................................13 Host Range and Symptoms.............................................................................................15 Disease Cycle..................................................................................................................16 Biology and Epidemiology..............................................................................................17 Genetics and Physiology.................................................................................................19 Detection and Diagnosis..................................................................................................19 Powdery Mildew Control................................................................................................22 Cultural methods and resistant varieties...................................................................22 Chemical control......................................................................................................23 Alternative measures................................................................................................24 Induced resistance....................................................................................................25 Biological control.....................................................................................................26 Oils...........................................................................................................................28 Inorganic chemicals: Potassium bicarbonate and phosphorous acid........................29 Silicon.......................................................................................................................31 3 EVALUATION OF SILICON FOR SUPPRESSING POWDERY MILDEW DEVELOPMENT IN GERBERA DAISY.............................................................................37 Introduction................................................................................................................... ..........37 Materials and Methods.......................................................................................................... .39 Effect of Calcium Silicate in Powdery Mildew Development in Gerbera Plants...........39 Effects of Silicon in Horticultura l Traits of Gerbera Flowers.........................................40 Evaluation of Silicon Accumulation over Time..............................................................40 Silicon Extraction from Gerbera Leaves.........................................................................41 Results........................................................................................................................ .............42 Effect of Silicon on Powdery Mildew Development in Gerbera Plants..........................42 Effects of Silicon in Horticultura l Traits of Gerbera Flowers.........................................42 Evaluation of Silicon Accumulation 2, 5, 9, 16 and 23 Days after Transplant...............43
6 Discussion..................................................................................................................... ..........44 4 EVALUATION OF BIOFUNGICIDE PR ODUCTS FOR MANAGING POWDERY MILDEW IN GERBERA DAISY..........................................................................................54 Materials and Methods.......................................................................................................... .55 Results........................................................................................................................ .............56 Discussion..................................................................................................................... ..........58 LIST OF REFERENCES............................................................................................................. ..69 BIOGRAPHICAL SKETCH.........................................................................................................83
7 LIST OF TABLES Table page 3-1 Effect of amending potting so il with calcium silicate (CaSiO3) on powdery mildew of gerbera daisy............................................................................................................... ...50 32 Silicon (Si) content in ne w leaves of gerbera daisy...........................................................51 3-3 Effect of amending potting so il with calcium silicate (CaSiO3) in horticultural traits of gerbera flowers............................................................................................................. .51 3-4 Analysis of covariance for silicon cont ent in gerbera leaves at 2, 5, 9, 16, and 23 days after transplant (DAT)...............................................................................................52 3-5 Silicon content in gerbera leaves at 2, 5, 9, 16, and 23 days after transplant (DAT)........52 3-6 Effect of silicon on powdery mildew seve rity in gerbera leaves. Experiment I................53 3-7 Effect of silicon on powdery mildew seve rity in gerbera leaves. Experiment II...............53 4-1 Source, rate, active ingredient, and manuf acturer of biofungicide s used to suppress powdery mildew in gerbera daisy......................................................................................64 4-2 Effect of biofungicides and conventional fungicides on powdery mildew severity in gerbera daisy.................................................................................................................. ....65 4-3 Effect of treatments on powdery mildew severity in gerbera cultivars treated with biofungicides and conve ntional fungicides........................................................................66
8 LIST OF FIGURES Figure page 4-1 Daily average temperature and relative humidity in greenhouse from April to June, 2007........................................................................................................................... .........67 4-2 Disease progression of powdery mildew for the gerbera cultivars used in the biofungicide experiment....................................................................................................67 4-3 Area under disease progress curve (AUDPC) values for se verity of powdery mildew in gerbera daisy treated with biofungicide s and conventional fungicides. Bars with the same letter in each cultivar do not differ significantly according to Fishers protected LSD (P 0.05)...................................................................................................68
9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF SILICON AND BIOFU NGICIDE PRODUCTS FOR MANAGING POWDERY MILDEW CAUSED BY Podosphaera fusca IN GERBERA DAISY ( Gerbera jamesonii ) By Catalina Moyer August 2007 Chair: Natalia A. Peres Cochair: Lawrence E. Datnoff Major: Plant Pathology Gerbera daisy ( Gerbera jamesonii Bolus ex. Hook f.) is an ornamental plant grown in Florida under greenhouse or shade house conditions as potted plan ts and for landscape. Gerbera plants are highly susceptible to powdery mildew caused by the fungus Erysiphe cichoracearum DC. or Podosphaera (Syn. Sphaerotheca ) fusca (Fr.) S. Blumer. This disease affects all plant parts and reductions in yield and in quality are the main components of economic loss. Environmental conditions of high humidity a nd moderate temperatures, which are highly prevalent in Florida, are the most conducive fo r powdery mildew development. Consequently, most nurseries use repeated applications of f ungicides as the main method for powdery mildew control. However, pathogens may develop re sistance to some fungicides after consecutive applications and thus, other alte rnatives to powdery mildew c ontrol are in demand. The objective of this study was to evaluate the efficacy of silicon and other biofungicides for suppressing powdery mildew in gerbera daisy. The effect of s ilicon as calcium silicate, incorporated into the growing medium, or potassium silicate, applie d as a drench, was evaluated in the highly susceptible cultivar Snow white. The effect of silicon in flower quality and the silicon content in gerbera leaves was determined as well. The e ffect of spray applications of the biofungicide
10 products Actigard (acibenzolar-S-methyl), Kphite (phosphorous acid), Milstop (potassium bicarbonate), Prevam (boron, orange oil and organic surfactants), Rhapsody ( Bacillus subtilis ) and AgSil (potassium silicate) was evaluated in highly susceptible (Snow White and Orange) and moderately susceptible (Hot Pink and Fuc hsia) cultivars. Powd ery mildew caused by Podosphaera fusca developed in the plants from natural inoculum. Disease severity was assessed and ratings were used to calculate the area under disease progress curve (AUDPC). Results suggested that neither calcium silicate nor pot assium silicate were effective in suppressing powdery mildew in gerbera daisy. These silicon sources did not have an effect on flower quality nor did they accumulate in gerbera leaves. Th e biofungicides products Actigard, Agsil, K-phite, Milstop, Prevam, and Rhapsody suppressed powdery mildew of gerbera daisy compared with untreated plants; however, these products were not as effective as the fungicide program of Heritage alternated with Eagle. Among the bi ofungicides tested, Actigard and Agsil were the least effective treatments. Rhapsody provided m oderate disease control and K-phite, Millstop and Prevam were the most effective in reducing disease severity. The level of disease reduction obtained with Prevam in cultivars Fuchsia, Hot pi nk and Orange compared to that attained with the systemic fungicides. In conclusion, biofungi cide products can signif icantly reduce powdery mildew compared to no treatment and products su ch as Prevam, K-phite, and Millstop may be used in alternation with system ic fungicides as part of an integrated disease management program and as an alternative to reduce the use of fungicides fo r suppressing powdery mildew of gerbera daisy.
11 CHAPTER 1 INTRODUCTION In 2005, the wholesale value of be dding and garden plants in th e US increased four percent giving this industry a total valu e of about $1.5 billion. Bedding a nd garden plants are mainly produced in California, Florida, Michigan, New York, Ohio and Texas. The leading producers of potted flowering plants are Califor nia and Florida. Florida has th e largest floriculture production area with 15, 900 acres and about 10 % of this area is in perman ent greenhouses; the remainder is either in the open field or under shade or temporary cover. In 2 005, floriculture sales in Florida from potted flowering plants reached $95 million (USDA, 2006). Gerbera daisy ( Gerbera jamesonii Bolus ex. Hook f.), belonging to the family Asteraceae, is an evergreen perennial grown as an ornament al plant. It produces flowers borne in stems arising from the crown, and it is commercialized as cut flowers, potted plants or for landscape (Tija and Black, 2003). Gerbera da isy originated in South Afri ca and were introduced to the Americas in the 1920s.In 1932 gerberas were cu ltivated outdoors in Florida (The gerbera association). Nowadays, gerberas in Florid a are produced mainly under greenhouse or shade house as potted and bedding plants. Gerberas are propagated by seed and vegeta tively through tissue culture. Propagative material is produced both by US growers (Florida leads this industry) and in Central America (Costa Rica, Guatemala and Mexico). Immature pl ants and seedlings are started abroad and are brought into the US to mature further in nur series prior to fina l sale (USDA, 2006). Gerberas are susceptible to several pests a nd diseases. Powdery mildew is an important fungal disease in gerberas and it can be caused by two species, Erysiphe cichoracearum DC and Podosphaera (Syn. Sphaerotheca) fusca (Fr.) S. Blumer (Daughtrey et al., 1995). This disease
12 affects all plant parts and reductions in yield and in quality are the most important components of economic loss. Environmental conditions most conductive for powdery mildew development include high humidity (80% to 90%) and moderate temperatures from 20 to 28C (Daughtrey et al., 1995); these conditions are prevalent in Florida. Standard practices for powdery mildew contro l include chemical, cu ltural and biological methods (Agrios, 2005). Nurseries in Florida ma inly control powdery mildew through repeated applications of fungicides (Lar son and Nesheim, 2000). However, consumers perception of the impact of pesticides on the environment and human health has intens ified the search for alternative methods of disease control (Gullino et al., 1999). In recent years, several alte rnative products including biologic al control agents, potassium bicarbonate, phosphorous acid, compounds that induce systemic resist ance, oils and silicon have been reported to control powdery mildew. Thes e products are often described as biofungicides and they can directly affect fungal organisms or may stimulate the plants defense mechanisms (McGrath, 2004). The goal of this study is to evaluate the efficacy of silicon and several biofungicide products for the management of powdery mild ew in gerbera daisies grown under greenhouse conditions in Florida.
13 CHAPTER 2 LITERATURE REVIEW Powdery Mildew Powdery mildew describes the organism as well as the disease caused by a group of fungi that are obligate parasites or biotrophs meaning th at they can only grow and multiply in a living organism (Agrios, 2005). As many other fungi, the causal agent of pow dery mildew has two names and the names are based on its reproductive stage. The teleomorph ic or perfect stage describes a fungus when reproducing sexually an d anamorphic stage describes the fungus when reproducing asexually. The perfect stage refers to a fungus with wh ite hyphae and courless ascospores (sexual spores) formed within a sac known as ascus (pl. = asci) enclosed in an ascocarp (i.e., cleistothecia) th at is present on the surface of plant tissues at the end of the growing season (Heffer et al., 2006). The anamorph is the stage of the fungus that develops as white mycelium (mass of hyphae) on the surface of plant tissue, with one-celled conidia (asexual spore) produced singly or in chains at the tip s of conidiophores (specia lized hyphae) that arise from the mycelium (Agrios, 2005). Some species of powdery mildew are known to produce only conidia but many others produce co nidia and then clestothecia wh en environmental or nutritional conditions become favorable. Thus two names th at differentiate the two reproductive phases describe the same organism. However the sexual name is generally used even though the sexual stage is rarely found (Yarwood, 1978). Powdery m ildew of gerbera daisies is caused by two fungal species Erysiphe cichoracearum DC.and Podosphaera (Syn. Sphaerotheca) fusca (Fr.) S. Blumer (Daughtrey et al., 1995). Taxonomy and Phylogenetic History Linnaeus, in 1753, gave the first binomial name to the causal agent of powdery mildew as Mucor erysiphe based only on the white mycelial stage of the fungus. The name Mucor was
14 dropped since it had been used previously for another fungus similar to what is now called Rhizopus ; however, the specific epithet e rysiphe continued to be used as a genus and later as a family name (Yarwood, 1978). Todays taxonomy is based on the work of Leveille in 1851. He considered powdery mildew fungi as a single and separate family and intr oduced a classification system with a key for the genera. This system was based on the perfect stage of the fungus, and species were differentiated based on number of asci per ascocarp, number of ascospores per asci and the morphology of appendages; however, the importance of anamorphs was not recognized (Yarwood, 1978). Ten years later, the Tulasne br others recognized the connection between anamorphic and teleomorphic stages and assigne d the species causing powdery mildew to the family Erysiphaceae (Braun et al., 2002). Taxonomic research in powdery mildew is now based not only in morphological features but also on electron microscopy and molecular tools. The current classification is similar to the one pr oposed by Braun in 1987, but it has been changed based on recent molecular studies (Braun et al ., 2002). Saenz and Taylor (1999) studied the phylogenetic relationships among 45 powdery mild ews species plus two outgroup species using internal transcribed spacer (IT S) sequences and 17 morphological characters. Results from both morphological and molecular data revealed th at the powdery mildew agents formed six evolutionary lineages that include: Clade 1 Erysiphe Microsphaera and Uncinula ; Clade 2 Erysiphe galeopsidis and Erysiphe cumminsiana ; Clade 3 Erysiphe species; Clade 4 Leveillula and Phyllactinia Clade 5 Sphaerotheca Podosphaera and Cystotheca ; Clade 6 Blumeria graminis All clades have an asexual stage called Oidium except clade 4 for which the asexual states are known as either Oidiopsis or Ovulariopsis The species causing powdery mildew on gerber a daisies originated from two different lineages (clade 3 and5); both spec ies belonging to the class Ascomy cetes; order Erysiphales and
15 family Erysiphaceae. The species Erysiphe cichoracearum is grouped within the tribe Erysipheae, and the species, Podosphaera (Syn. Sphaerotheca) fusca within the tribe Cystotheceae, subtribe Cystothecinae (A grios 2005; Braun et al., 2002). The genus Podosphaera was introduced in 1823 (Braun et al., 2002). Furthe rmore, Takamatsu et al. (2000) studied the evolutionary history of the Podos phaeras tribe Cystotheca, and th eir molecular results suggested that the genus Sphaerotheca originated from a Podosphaera -like ancestor and that their evolution involved a change in host range from arbor to herb species. Analogous results were reported previously by Saenz and Taylor (1999) who pointed out that since molecular data showed that Podosphaera and Sphaerotheca s pecies did not form different clades and given that Podosphaera is the older name, all Sphaerotheca species could be changed to Podosphaer ,thus, Podosphaera (syn. Sphaerotheca) Host Range and Symptoms Powdery mildew is a worldwide disease and it affects many species of plants including cereals, grapes, ornamentals, tr ees, and vegetables (Agrios, 20 05). Some powdery mildew fungi have a wide host range whereas othe rs are host specific. For instance, Erysiphe Laveillula and Sphaerotheca are common on annual herbs whereas Microsphaera Phyllactini, Podosphaera and Uncinula occur on woody plants, and Erysiphe graminis is specific to grasses and cereals (Yarwood, 1978). Flowering potted pl ants that are especially su sceptible to powdery mildew include begonia, dahlia, gerbera, hydrang ea and poinsettia (Daught rey et al., 1995). Powdery mildew differs from most diseases in that it does not cause symptoms other that a slow decline (or killing) of plant parts and decrease in host productivity (Yarwood, 1978). However, the disease is easy to identify by its co nspicuous signs that first appear as spots or patches of white mycelium on upper and lower leaf surfaces that can also spread to shoots, stems, flowers and fruits. The spots gradually enlarge and coalesce as hyphae and conidia are
16 produced in abundance, forming a white powderlike mat (Agrios, 2005). On artichoke, onion, pepper and tomato, yellow patches and little white powdery growth are produced on the leaves (Davis et al., 2001). Severely inf ected leaves turned pale yellow, brown and then die. At the end of the growing season, cleistothecia appear singly or in groups on the mycelium. Those structures are spherical, thick-walled, small, and at first white, then or ange red, and finally black (Agrios 2005; Alex opoulos and Mims, 1979). Disease Cycle Powdery mildew fungi produces white myceliu m from which short and erect specialized hyphae called conidiophores develo p (Agrios, 2005). Oval or r ound-shaped conidia are produced in chains at the tip of each conidiophore (Kendr ick, 2000). Conidia are disp ersed by air current to other parts of the plant or to other plants of the same species. If conditions of temperature and relative humidity are favorable, the conidia germinate and infect the host plant (Agrios, 2005). The mycelium formed during previous cropping seas ons serves as the primary inoculum which infects new emerging leaves. Conidia produced from the primary inoculum bring about secondary infections making powdery mildew a polycyclic disease (H effer et al., 2006). Conidia are deposited on the host plant and adhe re to its surfaces by sticky exudates that are produced before germination and, according to Carver et al. (1999) these exudates contain several proteins that may play a role in the infect ion process. The process involved in the recognition of leaf surfaces by th e conidia is not well understood; however, it is thought that those interactions initiate the emergence of a germ tube (Green et al., 2002). As conidia germinate development of a germ tube starts wh ich elongates and penetrates the leaf epidermis (Agrios, 2005). Three functions are attributed to the germ tube: the attachment of conidia to plant surface, a means for the conidium to access the host water and establish if the contact surface is suitable for germ tube elongation and developmen t of appressorium (Green et al., 2002). The
17 germ tube differentiates into an appressorium from which a speciali zed hyphae, called the penetration peg, crosses the cuticl e and penetrates the host epiderma l cell (Green et al., 2002). If penetration is successful, the peg differentiates into haustoria, a feeding st ructure that develops within the plasma membrane and feeds mainly by extracting glucose from its host (Zeyen et al., 2002). The germ tube continues to grow and sp reads across the leaf surface producing mycelium and many haustoria (Agrios, 2005). Through hausto ria, the fungus establishs a biotrophic relationship with the host. This life cycle takes place with all powdery mild ew systems except for Blumeria graminis This species will produce two germ tubes but only one will form appresoria while the other serves as a reserve (G reen et al., 2002; Zeyen et al., 2002). When environmental conditions become unfa vorable, production of conidia slows down and the fungus may switch to the production of cleistothecia containing one or several asci (Alexopolus and Mims, 1979). The sexual spores or ascospores are produced within the asci. Ascospores are released when the new season or host tissue becomes available and the cycle starts over (Agrios, 2005). Po wdery mildew species may survive as mycelium or by production of cleistothecia (Jarvis et al., 2002). Biology and Epidemiology During the agricultural growing season, powdery mildew agents reproduce prolifically by asexual conidia. The development of an epidem ic depends on environmental conditions as well as on parasite and host factors (Agrios, 2005). A uni que set of environmenta l conditions such as temperature, humidity and light will determine conidial and as cospore formation, release and dispersal (Jarvis et al., 2002). For example, the re lease of conidia from apple, cucumber, rose and sweet peas is periodic with th e highest release occu rring at noon and gradua lly declining in the afternoon and early ev ening (Pady, 1969).
18 Conidia once produced remain viable for 7 to 8 days and may be dispersed by air currents (Zitter et al., 1996). The separation of coni dia from the mother colony may trigger the germination process since conidia do not germin ate when attached to the conidiophores (Green et al., 2002). Requirements of environmental cond itions vary among isolates in a specific host (Jarvis et al., 2002). Thus, the range of conditi ons for powdery mildew development are quite wide and include periods of low to high rela tive humidity (below 50% to near 100%) and temperatures between 10 to 28C (Daughtrey et al., 1995; Horst, 1983; Jarvis et al., 2002; Zitter et al., 1996). Powdery mildew is more preval ent in shady areas, which may be related to differences in temperature and humidity (Butt, 1978). Conidia of powdery mildew species have the ability to germinate in the absence of fr ee water. Moreover, water on leaf surfaces is detrimental to the development of powdery mildew (Jarvis et al., 2002). Furthermore, conidial germination varies depending on a particular sp ecies. Conidia produced singly germinate faster than conidia borne in chains. For instance, species of Erysiphe take about 8 h whereas those of Podosphaera take more than 10 h to germinate. (Braun et al., 2002) The incubation period or time between infec tion and development of symptoms (i.e. colonies) is generally between 3 to 7 days (Butt, 1978; Zitter et al., 1996; Wheeler, 1978). However, the length of this period depends greatly on environmental conditions. Xu (1999) studied the effect of temperature on the roseSphaerotheca pannosa dynamics. He found that the shortest incubation period, 3 days, was at tained at temperatures between 22 and 24 C, whereas, the longest, 10 days, was reached at 8 C. Also, colonies expanded and had greater sporulation when temperatures were kept constant. Conseque ntly, he concluded that temperature influences the development of powdery mildew of rose by the number and size of colonies, rate of colony growth, and sporulation.
19 Genetics and Physiology With the exception of Blumeria graminis genetic and molecular analyses of powdery mildew fungi are not well advanced compared to other plant pathogens, and this maybe due to their nature as obligate biotrophic fungi (Ferna ndez-Ortuo et al., 2007). Genetic studies using molecular biological techniques have been conducted on powdery mildew fungi not only to study it s phylogeny but also to examine the distribution range of the fungus among plants. For instance, Hirata et al. (2000) showed that molecular data were consistent with the groupings of Sphaerotheca species based on infectivity. Isolates with a wide host range that co-infected acalypha, cosmos, cu cumber, zucchini, sunflower and zinnia, had identical nucleotide sequences, whereas other isol ates that infected marigold and gerbera were unable to co-infect and belong to different haplotypes. Moreover Hirata and Takamatsu (2001) reconfirmed the close relationship between phylog eny and infectivity of powdery mildew fungi. These authors stated that host specialization mi ght be what triggered genetic divergence among the powdery mildews. In a recent study, Fernandez-Ortuo et al. (2007) amplified the whole genome of Podosphaera fusca by using the amplification of phi 29 DNA polymerase-mediated multiple displacement amplification (MDA) technique, whic h allows the production of large amounts of DNA. They demonstrated that the amplificatio n of mitochondrial and nuclear fragments was identical whether using DNA genera ted from MDA or from the trad itional PCR-based technique. Thus, the MDA technique may faci litate molecular genetic studi es in powdery mildew fungi, which are very limited at present. Detection and Diagnosis Powdery mildew is probably the most easily recognizable plant disease (Agrios, 2005). Its conspicuous signs first appear as whitish colonies growing on livi ng leaves and are particularly
20 common in shady areas (Kendrick, 2000). The white growth of the fungus consisting of mycelium and conidiophores develops on both leaf surfaces and on shoots, stems, flowers and fruits (Davis et al., 2001). Disease signs usually develop first on older leav es and on some plants powdery mildew may cause distortion of the leav es (Daughtrey et al., 1995; Horst, 1983; Zitter et al., 1996). Identification of powdery m ildew fungi to genera and species used to rely on morphological characteristics of the se xual stage (Yarwood, 1978). Nowadays, new morphological and molecular approaches are used and isolates are differe ntiated not only by the sexual, but also by the asexual stage (Braun et al., 2002). An important characteristic for identification is whether conidia are produced in chains or singl y. However, this distinction can be difficult to observe, and in so me genera, particularly in the Erysipheae conidia that are produced singly can stick together to form ps eudochains. Other charac teristics that aid in classification are host specificity and the location of mycelium whether superficial (epiphytically) or within the leaf tissue (endophy tically) (Heffer, 2006). Cook et al. (1997) used light and scanning electron microscopy and host range data to identify and classify sixty powdery mildew f ungi in their anamorphic stage. Observations by light microscopy identified isolates based on size and shape of conidia, presence of fibrosin bodies, conidia in chains, patterns of outer wall of wrinkled conidia, char acteristics of mycelium and the degree of lobbing of appressoria. Obse rvations by scanning electron microscopy allowed more accurate identification by distinction of patte rns on the conidial surfaces such as the end walls separating conidia from conidiophores or from other conidia, and the outer walls of turgid and wrinkled conidia. Despite all characteri stics measured, some di screpancies were found among isolates and thus host range data were needed for identification.
21 In the case of gerbera daisies, the fungi that cause powdery mildew belong to two separate taxa with different morphological characteristics. For instance, Podosphaera have conidia with fibrosin bodies and crenate edge lines and, the germ tubes originate from the sidewall whereas Erysiphe does not have fibrosin bodies, the conidia have sinuate edge lines and the germ tubes originate from the end wall. These and other di fferences on the surface of the conidia make the two species relatively easy to recognize by using light and sc anning electron microscopy (Braun et al., 2002). Molecular techniques such as PCR-based analyses are often used for detection and identification of plant pathogens (Agrios, 2005). An accurate identification of plant pathogens is essential in plant quarantine and in locating th e origin and source of inoculum (Cook et al., 1997). Serological tests have also been used for the detection of powdery mildew fungi. Van Roestel et al. (1991) used polyclonal antibodies to detect the location of antigens of the fungus on the surface of germ inated conidia of Erysiphe graminis on barley. Results suggested that some antigens can be detected on the host before penetration by the fungus a nd that the levels of antigens increased as disease progressed. Studies on powdery mildew are difficult because of the obligate, biotrophic nature of the fungus that makes storage and propagation of isolates unpractical and time-consuming. Traditionally, conservation of powdery mild ew fungi is done by keeping the fungus on a susceptible host, and frequent transfers of c onidia onto fresh plant ti ssues. However, PerezGarcia et al. (2006) recently developed a method for long-term preservation of P. fusca. After desiccating conidia with silica gel from 6 to 12 h at 22C and keeping it at -80C for three years, they obtained a recovery of 100% After 4 to 5 years, the r ecovery decreased 20-25%. When
22 phenotypic and genetic stabilit y essays were conducted, no di fferences were found among the isolates before and after storage. This preservation t echnique may be very useful for diagnosis, fungicide resistance, po pulation genetics, or taxonomy st udies in powdery mildew fungi. Powdery Mildew Control Management of powdery mildew can be achieve d by using a variety of strategies such as cultural practices, resistant cultivars, use of ch emical products, sprays of defense activating compounds, biocontrol agents, salts and oils (A grios, 2005; Daughtrey et al., 1995; Malathrakis and Goumas, 1999; Horst, 1983; Zitter et al., 1996). Cultural methods and resistant varieties Cultural practices include planting in sunny areas, providing good air ventilation, and avoiding excessive fertilization (Agrios, 2005; Daughtrey et al., 1995; Horst, 1983). Overhead irrigation may reduce powdery mildew as it washes off conidia from the surfaces of leaves, but, it may contribute to other disease problems (Davis et al., 2001). Control of powdery mildew in cereals and in other annual crops is mainly achieved by the use of resistant cultivars (Agrios, 2005). Hsam and Zeller (2002) stated that, to date, diverse choices of resistance genes, gene combinations and modes of inheritance are available for breeding wheat cultivars resistant to powdery m ildew. Resistant cultivars are also used to manage powdery mildew of cucurbits and roses (Horst, 1983; Zitter et al., 1996). Fully resistant cultivars of melon and partially resistant cultivars of cucumber, pumpkin, squash, and watermelon are available (Malathrakis and Go umas, 1999; Jahn et al., 2002). However, disease resistant cultivars are not widely develope d for greenhouse crops (Elad et al., 1999) and, moreover, genetic resistance is not common in ornamental crops (Jarvis, 1992). Research on development of gerbera daisy cultivars resistant to powdery mildew is currently underway. In a recent study, Kloos et al. (2005) examined the rela tionship between the high level of resistance
23 of Gerbera hybrida to powdery mildew and trichome de nsity as they thought there was a relationship between those phenot ypes. Although they found that two major genes controlling those traits were unrelated, they provided preliminary informa tion for future powdery mildew resistance research on G. hybrida. Chemical control Sulfur has been used as treatment to contro l powdery mildew since 1802. It is inexpensive and relatively effective (Bent, 1978). In the ea rly 1990s, systemic fungi cides were developed (Agrios, 2005). Currently, fungicides to c ontrol powdery mildew include the group of strobilurins (QoI fungicides) su ch as trifloxystrobin sold as Flint (Bayer CropScience, North Carolina USA); azoxystrobin as Quadris, Herita ge or Abound (Syngenta, Greensboro, NC); and pyraclostrobin sold as Headline( Basf corporation, Florham Par k, NJ) or Heritage (Syngenta, Greensboro, NC). Other fungicides used are in the triazole group, such as myclobutanil sold as Nova or Eagle (Dow AgroSciences, Indianapol is, IN) (Agrios, 2005; Crop Data Management Systems, 2007). Inorganic chemicals such as sulfur and copper compounds are often used; however, phytotoxicity has occu rred (Nuez-Palenius, 2006). In the floriculture industry, gr owers rely on synthetic chemical s for the control of pests and diseases. However, chemical cont rol is not always completely effective, as some chemicals may no longer work due to the development of re sistance by pests and pathogens (Gullino and Wardlow, 1999). The risk of development of re sistance increases when a pathogen undergoes many and short diseases cycles per season (B rent, 1995). Systemic fungicides used for the management of powdery mildew have a high risk of developing resistance and powdery mildew has a high potential for development of resistance (McGrath, 2004; Brent, 1995). Resistance of powdery mildew has already occurred with the fungicides benomyl (Benlate, DuPont, Atlanta GA) and triadimefon (Baylet on, Bayer CropScience, North Carolina USA)
24 (McGrath, 2001). Studies to de termine the sensitivity of Uncinula necator the causal agent of powdery mildew on grapes, to strobilurin f ungicides were conducted in California and New York, and both studies agreed that powdery mild ew isolates have alr eady developed a higher tolerance to strobilurin fungi cides (Miller and Gubler, 2004; Wong and Wilcox, 2002). Also, in Japan, azoxystrobin failed to control Podosphaera fusca in cucumber, and a fungicide sensitivity tests and DNA sequence analysis confirmed the development of resistance to strobilurin fungicides (Ishii et al., 2001). Furthermore, th e failure of myclobutan il to control powdery mildew in grapevines was confirmed in Ca nada in 2000 (Northover and Homeyer, 2001). A successful disease management program must involve strategies for resistance management to delay the build-up of resi stant strains (McGrath, 2004). The Fungicide Resistance Action Committee (FRAC) provides fungicide resistan ce management guidelines to prolong the effectiveness of "at risk" fungicides. The strate gies for resistance management include: 1. Use of mixtures of pr oducts with one or more fungi cides with different modes of action or different chemical groups, or use as one component in a rota tion or alternation. 2. Restriction of the number of tr eatments applied per season, and application only when strictly necessary. 3. Use of products at the manufacturer's recommended ra te. 4. Avoidance of systemic fungicides. 5. Use IPM (Integrated Pe st Management) (Brent, 1995). Alternative measures The demand by consumers for high plant quality forces growers to intensify their spray programs against diseases. This practice may l ead to the faster development of pesticide resistance while increasing overa ll production costs. However, c onsumer awareness of potential effects of pesticides on the environment and human health has intens ified the search for alternative methods of disease control (Gullino et al, 1999). Bi ofungicides are certain types of naturally based microbial or biochemical produc t derived from animals, plants, bacteria, and
25 certain minerals. These products can affect f ungal organisms directly or may stimulate the plants own defense. They are generally narro w-spectrum, decompose quickly, and thus are considered to have low potential for nega tive impact on the envi ronment. In addition, biofungicides require less data for registration and are approved in faster than conventional pesticides (McGrath, 2004; EP A, 2007). Biofungicides products such as Actigard, biological control agents, oils, potassi um bicarbonate, phosphorous aci d, and silicon, have been successfully used against powdery mildew (Belanger et al., 1 994; Belanger and Labbe, 2002; Ghanmi et al., 2004; Grove et al., 2005; McGrath and Shish koff 1999; Menzies et al., 1992; Pasini et al., 1997; Reuveni et al., 1996). Induced resistance In nature, most plants defend themselves agai nst pathogens by the effect of constitutive barriers or by activating protective mechanis ms, a processes known as induced resistance (Sticher et al., 1997). Defense responses are a se t of metabolic alterations that not only accumulate locally at the site of infection but are also induced systemically, such as the development of systemic acquired resistance (SAR ) or induced systemic resistance (ISR) that prevents further attacks from fungi, bacteria and viruses (Vallad a nd Goodman, 2004; Van Loon and Van Strien, 1999). Defense mechanisms that are elicited include rein forcement of cell wall by papilla formation, callose and lignin depositio n, production of antimicrobial phytoalexins or phenolic compounds and synthesis of antifungal proteins such as 1,3 glucanases and chitinases (Belanger and Labbe, 2002; Salmeron et al., 2002). SAR can be triggered by microbes or by natural or synthetic chemicals such as salicylic acid (SA), 2,6-dichloro-isonicoti nic acid (INA) or benzo(1,2,3)thi adiazole-7-carbothionic acid Smethyl ester (BTH). The latter has been marketed as Actigard, Bion and Boost. In contrast, ISR is activated by rhizobacteria such as several strains of Pseudomonas (Vallad and Goodman,
26 2004). When SAR is activated, salicylic acid (SA) and pathogenesis related proteins accumulates at the site of infection an d moves throughout the plant (M auch-Mani and Metraux, 1998). Products such as Actigard, potassium s ilicate, and microorganims such as Bacillus subtilis have been reported to induce resistance agai nst powdery mildew (Belanger and Labbe, 2002). Actigard alone was effective in reducing the in cidence of powdery mildew in several lettuce cultivars (Matheron and Porcha s, 2000); however, alternating Actigard with commercial fungicides resulted in high levels of powdery mildew suppression in lettuce (Matheron and Porchas, 2004). Moreover, Actigar d was effective against powder y mildew in cabbage and in muskmelon (Matheron and Porcha s, 2003; Miller and Hernandez, 2001). However, Actigard did not provide a satisfactory level of disease reduction in pumpkin (Babadoost, 2002). Moreover, it was ineffective when used as a protectant agai nst powdery mildew of cucumber suggesting that it failed to enhance the defense responses in this plant (Wurms et al., 1999) Therefore, activation of resistance is independent of the target pathogen and it may be determined by the crop (Oostendorp et al., 2001). Biological control The term biocontrol that is used in plant pathology en tails the use of antagonistic microorganisms to suppress plant diseases. The or ganisms that suppress pe sts or plant pathogens are referred to as the biological control agen ts (BCA) (Agrios, 2005; Pal and McSpadden, 2006). The mechanisms involved in biological control include antibiosis, competition, cross protection and parasitism (Jarvis, 1992; Shoda, 2000). BCA have been used to control powdery mildew since the early 1900s, and their us e has increased in the last 20 years especially under greenhouse conditions (Belanger and Labbe, 2002). There are approximately 40 fungal species that have been reported or have been tested as prospect ive biocontrol agents of powdery mildew (Kiss, 2003). Several BCA have been registered for the control of powdery mildew and it is presumed
27 that the interaction of these organisms with powdery mildew is not limited to one mode of action. Ampelomyces quisqualis (AQ10) colonizes the hyphae and conidiophores of its host. Tilletiopsis spp, Pseudozyma flocculosa (Sporodex, Plant Products Co., Brampton, Ontario, Canada) and Bacillus subtilis QST 713 (Serenade or Rhapsody, AgraQuest, Inc. Davis, CA), act by antibiosis meaning that they first kill or weaken their host through the release of antibiotics and then attack the structures of the host fungus (Pauli tz and Belanger, 2001). Moreover, B. subtilis is capable of inducing disease resistance in some crops (Belanger and Labbe, 2002). The major constraint to BCA is th eir requirement for high humidity. The efficacy of biological control for powdery mildew on roses varied with the level of relative humidity prevalent in the greenhouse; however, the additi on of 1% paraffin oil to the spore suspension reduced the dependency of high hu midity and improved the activity of the BCA (Belanger et al., 1994). Ampelomyces quisqualis was as effective as the commer cial fungicide dodemorph in controlling rose powdery mildew When the BCA was used in rotation with other antifungal compounds, the efficiency increased to a higher le vel than the control pr ovided by the fungicide alone (Pasini et al., 1997). In a similar study, P flocculosa was used as the BCA for powdery mildew control on roses. The anta gonist fungus was as effective as the registered fungicide and only seven applications were suffi cient to attain a satisfactory level of control compared to 10 sprays of the fungicide dodemorph acetate (M eltatox, BASF Corporati on, Florham Park, NJ) (Belanger et al., 1994). Moreover, the bacteria Bacillus spp. provided an 80% reduction in powdery mildew severity of cucumber caused by P. fusca as determined by in vitro studies with detached leaves and seedli ngs (Romero et al., 2004).
28 The BCA Bacillus subtilis has also been shown to reduce di sease severity ina a number of host-pathogen systems including Diaporthe citri on grapefruit (Agostini et al., 2003); rust caused by Uromyces appendiculatus on snap beans (Baker et al., 1985); brown rot caused by Monilinia fruticola on stone fruit (Pusey and Wilson, 1984), and stem rot caused by Fusarium roseum on carnations (Aldrich and Baker, 1970). Strains of Bacillus are resi stant to adverse environmental conditions and are viable for several years unde r storage conditions, making them advantageous over other BCAs (Shoda, 2000). Oils Different types of oils but mostly mineral a nd plant oils are capabl e of controlling plant diseases (Calpouzos, 1966). Oils derived from petrol eum or from seeds of plants have been used for control of diseases such as leaf spots and powdery mildews. They have not only been effective but also useful in reducing the develo pment of resistance to f ungicides (Agrios, 2005). Presumably, oils have four modes of action: prop hylactic, curative either be fore or after visible infection and antisporulative (McGrath and Shishkoff, 2000; Northover and Schneider, 1996). Early studies using mineral oils to control diseases are thos e reviewed by Calpouzos (1966) where oils were used as fungicides for controlling leaf spot on banana, grea sy spot of citrus and other hosts, late blight on celery, rust dise ases as well as downy and powdery mildews of cucumber. More recently, reduction of powdery m ildew severity has been reported on apple, cherry, cucurbits, grapes and roses. The levels of disease reduction range d from highly effective (McGrath and Shishkoff, 1999, 2000; Pasini et al., 1997) to slight ly satisfactory (Fernandez et al., 2006; Grove et al., 2005). And, in some cases, the efficacy of oils to reduce powdery mildew was comparable to those obtained with standa rd fungicides (Northover and Schneider, 1996) or even superior (Grove et al., 2000; Wojdyla, 2002).
29 Prevam, a blend of boron, orange oil and organi c surfactants provided effective control of powdery mildew in strawberries (Mertely et al., 2005). Boron is a micronutrient for vascular plants and it is thought to be involved in vari ous metabolic pathways such as lignification, cell wall structure, and phenol metabolism, among othe rs (Marschner, 1995). Ou r preliminary studies indicated that Prevam provided satisfactory c ontrol of powdery mild ew in gerbera daisy (unpublished data). The oil ingredient in Prev am breaks down fungal mycelia and spores and exposes them to desiccation and th us prevents further infection. Inorganic chemicals: Potassium bi carbonate and phosphorous acid Phosphonate and carbonate compounds such as phosphorous acid, sodi um bicarbonate and potassium bicarbonate, are used in plant disease management (Agrios, 2005). Certain inorganic compounds can induce systemic acquired resistance (SAR) in plants (Sticher et al., 1997). Phosphorous acid (H3PO3) is the active ingredient in phos phate or phosphonate and these products are used in agriculture for disease control or as a source of plant phosphorus (P) nutrition; however H3PO3 is not a nutritional sour ce of P for plants and it is often confused with phosphoric acid (H4PO4)(Brunings et al., 2005; McDonald et al., 2001). Phosphite based products are marketed under the trade names AGR I-FOS, BioPhos or Vital (Agrisel USA, Inc., Brookfield, CT ), Aliette or Fosetyl-Al (Bay er CropScience, North Carolina USA), K-phite (Plant Food Systems, Inc. Zellwood, FL), Pr oPhyt (Luxembourg industries Ltda., Tel Aviv, Israel), and Resist 57 (Act agro LLC., Biola, CA). H3PO3 products have been used to control soilborne diseases caused by oomycota (Brunings et al., 2005). However, limited information is available about their effectiveness against ot her plant pathogens (H eaton and Dullahide, 1990). Phosphite products have an antifungal activity against Phytophthora species (Fenn and Coffey, 1984). Guest and Grant (1991) proposed that phosph ites act both directly on the pathogen and
30 indirectly in stimulating host defense responses Regardless, phosphite is most effective when applied prior to infection (Jackson et al., 2000; Marks and Smith, 1992). Phosphite reduced the incidence of Phytopht hora root and crown rot on green peppers grown hydroponically (Frster et al ., 1998). Furthermore, since ProPhyt was as effective as the synthetic fungicides : Abound, Ridomil Gold (Syngenta, Greensboro, NC) and Cabrio (BASF Corporation, Florham Park, NJ) for the control of strawberry leather rot, it was suggested that alternating fungicides with phosphi te should provide effective cont rol of leather rot as well as reduce the risk of development of fungicide resistance (Rebollar-Alviter, 2005). In addition, foliar sprays of Aliette, were effective in redu cing disease severity caused by scab, melanose and brown spot on citrus (Agostini et al., 2003). There are few reports of effectiveness of phosphite products to decrease severity of powdery mildew Schilder et al. (2003) reported that ProPhyt reduced powdery mildew incidence and severity on grapes equivalent to the reduction provided by commercial fungicides. Similarly, Fosphite was effective in reducing powdery mildew on muskmelon when compared with untreated plan ts (Matheron and Porcha s, 2005). Furthermore, foliar sprays of Biophos reduced severity of E. cichoracearum on gerbera daisy (Mueller et al., 2003a). Some potassium bicarbonate-based products are label as fungicides and have been approved by the U.S. Environmental Protec tion Agency (EPA) (Belanger and Labbe, 2002). Armicarb 100 (Helena Chemical Company, Collierville, TN), Kaligreen(AgBio Inc., Westminster, CO), MilStop (BioWorks, Inc. Fa irport, NY), and Remedy (Bonide products, INC., Oriskany, NY) are all trade names for potassium bicarbonate based fungi cides and are labeled for powdery mildew control in conventional systems (Crop Data Management Systems, 2007) and organic crops (Kuepper et al., 2001). Kaligreen significan tly suppressed pow dery mildew
31 (caused by Sphaerotheca fusca ) on winter squash, muskmelon, and pumpkin compared with untreated plants (Matheron and Porchas, 2003; McGrath and Shis hkoff, 1999). Potassium bicarbonates were the most effective in reduci ng powdery mildew in cu cumber, roses, sweet peppers and tomato (Dik et al., 2003). In addition, folia r sprays of potassium bicarbonate applied preventively in combination with mineral oil we re very effective in reducing powdery mildew on cucumber and pumpkin (Ziv and Zitter, 1992). Furthermore, Kaligreen, Armicarb and Milstop have been used successfully to reduce powdery mildew of gerbera daisy (Sconyers and Hausbeck, 2004; Uchida and Kadooka, 2001). Othe r bicarbonate products such as sodium bicarbonate (NaHCO3) gave satisfactory control of pow dery mildew of grapevines, mango, nectarine and roses when applie d individually at weekly inte rvals or alternated with a commercial fungicide (Pasini et al ., 1997; Reuveni and Reuveni, 1995). In vitro studies showed that bicarbonate products have a fungicidal activity s uggesting that this may be the mode of action against plant pathoge ns (Punja and Grogan, 1982). Silicon Silicon (Si) in nature is found co mbined with oxygen to form SiO2, which is the simplest source of silica, and is the second most abundant element in the earths crust (Epstein, 1999; Schottman, 1979). Quartz reacts with water and forms silicic acid (H4SiO4) in which Si is found in the soil solution at concentrations between 0.1 to 0.6mM (Epstein, 1999, 1994). Some plants are capable of absorbing Si from the soil soluti on between a pH of 4 to 9. Following absorption, silicic acid is translocated to the shoots and, as water is lost by transp iration, amorphous silica gel, SiO2.H2O is deposited in cell walls, mainly in ep idermal and vascular tissues (Ma et al., 2001; Russell, 1988). Si content in plant tissue can range from 0.1 to 10% on a dry weight basis (Epstein, 1994).
32 Ma and Takahashi (2002) differentiated Si-accumulating and non-accumulating plants based on Si content and Si/Ca rati o. Plants with a Si content hi gher than 1% and a Si/Ca ratio higher than 1 are defined as Si accumulators. Inte rmediate accumulators are those plants that contain 0.5%-1% Si and the Si/Ca ratio is less than 1. Non-accumulators or Si excluders are plants that have less than 0.5% Si and a Si/Ca ratio lower than 0.5. In a recent study, Hodson et al. (2005) examined the phylogenetic variation of Si content on leaf or shoot tissues among 735 plant species and concluded that Si concentra tion was low in angiosperms, gymnosperms and ferns compared to non-vascular plant speci es (mosses and algae) and horsetails. Among plant species cropped for human cons umption, cucumber, gr ape, maize, rice, strawberry and wheat have the cap ability of Si uptake (Belanger et al., 2003; Bo wen et al., 1992; Kanto et al., 2007; Liang et al., 2006; Mitani and Ma, 2005). Currentl y, the gene that controls Si accumulation has only been characterized for rice. Ma et al. (2006) identified the low Si rice 1 (Lsil1) gene that was expressed mainly in rice root s. Lsil1 is the Si transporter gene in rice and was localized at the distal side of plasma membrane of both exodermal and endodermal cells. Solutes are unable to cross the pl asma membrane and transporters are needed for translocation of solutes from roots to shoots (Marschner, 1995). Si has been reported as a be neficial element in plants (Havlin et al., 2005; Marschner, 1995; Mengel and Kirkby, 1987) as it affords protect ion against biotic and abiotic stresses (Ma et al., 2001). A recent study demonstrated that Si could enhance the growth and appearance of flowering ornamentals. Savvas et al. (2002) reported that gerbera plants supplied with Si in the nutrient solution had significantly thicker flower stems and a highe r proportion of flowers graded Class I. Moreover, Richter (2001) revealed that vase life of different gerbera cultivars can be increased by supplying plants with Si and thus reducing the number of flowers with bent neck.
33 Si has been shown to reduce plant diseases in a number of plants including arabidopsis, avocado, barley, Bermuda grass, coffee, cucu mber, grape, muskmelon, rice, strawberry, sugarcane, St. Agustine grass, wheat, zucchini an d flowering ornamentals such as poinsettia and rose (Bekker et al., 2005; Belanger et al., 2003; Bo telho, et al., 2005; Bowe n et al., 1992; Brecht et al., 2004; Cherif et al., 1992a,b; Datnoff et al., 2005; Ghanmi et al., 2004; Gillman et al., 2003; Kanto et al., 2006; McAvoy and Bible, 1996; Menz ies et al., 1992; Remus-Borel, 2005; Seebold et al., 2001; Wiese et al., 2005). However, most Si research has been conducted on rice and cucumber. Datnoff et al. (1997) reported th at calcium silicate applied to Si-deficient organic soils in south Florida resulted in signifi cant reductions of two foliar funga l diseases of rice, blast and brown spot. Moreover, Si provided disease control as effective as that of synthetic fungicides in rice and turf (Brecht et al. 2004; Seebold et al., 2004) suggesting that fungicide rates could be reduced when used in combination with Si (See bold et al., 2004). Furthermore, when Si was applied to susceptible and partially resistant ri ce cultivars, the level of resistance was increased to that of the resistant cultivar (Rodrigue s et al., 2001; Seebold et al., 2001, 2000). Si affects the parasitic fitness of different fungi. Seebold et al. (2001) examined the effect of different rates of Si on the components of re sistance to rice blast. Wh en the rate of calcium silicate was increased, the incubation period was longer and the number of sporulating lesions decreased for the susceptible and partially resist ant cultivars. Similarly, Si affected several components of resistance of cucumber s to powdery mildew caused by S. fuliginea Colony number, colony area and percenta ge of conidial germination were reduced by increasing Si amendments from 0.5 to 2.3 mM (Menzies et al., 1991a). In addition, foliar or root applications
34 of potassium silicate reduced the number of co lonies of powdery mildew fungi on cucumber, muskmelon and zucchini squash (Men zies et al., 1992) and on grape leaves (Bowen et al., 1992). Fungal development on Arabidopsis thaliana was limited and rarely observed when plants were watered with a nutrient solution contai ning soluble Si (Ghanmi et al., 2004). In Japan, liquid potassium silicate was effective in suppressing powdery mildew on a susceptible strawberry cultivar Toyonoka in soil as well as in hydroponic cultivation (Kanto et al., 2004, 2006). Moreover, Belanger et al. (2003) found that Si amendments protected wheat plants from powdery mildew caused by Blumeria graminis f.sp. tritici Si has also been reported to control other diseases Black spot infection of Rosa hybrida Meipelta was suppressed by the addition of 150 mg/L Si as potassium silicate to irrigation water (Gillman et al., 2003). In vitro effect of soluble Si in several plant pathogenic fungi revealed that Si inhibited growth of Alternaria, Curvularia, Fusa rium, Phytophthora, Pythium, Stemphylium and Verticillium species at concentrations between 5 to 20 ml of Si per liter of agar suggesting that Si has selective fungicidal prope rties (Kaiser et al., 2005 ). Nevertheless, this contradicts the results found by Bowen et al. (19 92), Menzies et al. (1992) and Kanto et al. (2007) as their in vitro assays demonstrated that conidial germ ination and germ tube elongation of powdery mildew fungi was unaffected by potassium silicate. Currently, there are two running hypothesis on how Si can enhance resistance of plants to diseases. Initially, the protective ro le of Si was attributed to the accumulation of Si in the leaves creating a physical barrier to pathogens (Adatia and Besford, 1986; Epstein, 1994; Samuels et al., 1991a,b; Raven, 1983). However, recen t studies indicate that Si has a more active role in protecting plants against diseases by inducing th e plants own defense mechanisms (Fauteux et al., 2006; Fawe et al., 1998; Remus-Borel, 2005; Rodr igues et al., 2004). So, th e role of Si is not
35 solely limited to a physical barrier since Si also induces metabolic ch anges in Sifertilized plants (Epstein, 1999; Faut eux et al., 2006). Because of Si deposition in plant cell walls, it is expected that Si strengthens plant tissues and thus creates a physical barrier that impede s or slows down penetra tion of plant pathogens (Ma et al., 2001). Adatia and Besf ord (1986) reported anatomical and morphological effects of Si on cucumber leaves as they had a rougher texture, were more erect, with a darker color and their senescence was delayed. Bowen et al (1992) examined the effect of root and leaf applications of soluble Si on powdery mildew severity on grapes. Reduction of disease severity was attributed in part to a physical barrier created by thick potassium silicate deposits on the leaf that limited hyphal penetration. However, they also considered the role of Si in inducing a resistance response because of the movement of Si and its de position at fungal penetrat ion sites. Kim at al. (2002) investigated the location of Si in ri ce leaves by electron microscopy and X-ray microanalysis. Their findings confirmed that Si was deposited in epidermal cell walls, middle lamella and intercellular spaces of rice leaves providing a physica l barrier for the penetration and invasion of the fungus Magnaporthe grisea Cherif et al. (1994) reported th at supplying cucumber plants with Si produced an intense activation of peroxidases (POD) polyphenoloxidases (PPO) and chitinases after infection with Pythium spp. Similar results were found by Fawe et al. (1998) who demonstrated an enhanced resistance to Sphaerotheca fuliginea in cucumber by increase d antifungal activity from production of phytoalexins. Similarl y, Liang et al. (2005) found that root-applied Si enhanced the activity of pathogenesis-related (PR) proteins and thus increased re sistance to pathogen attack on cucumber plants.
36 The mechanism of induced resistance has been extensively studied in other pathosystems. Infection of wheat plants by Blumeria graminis was reduced by Si amendm ents and this response was associated with specific defense reactions (callose and papilla formation, deposition of chitin, B-1,3 glucans and phenolic compounds) found around fungal cell walls, haustoria and epidermal cell walls (Belanger et al., 2003). Si milarly, reduction of powdery mildew of wheat was attributed to the presence of phenolic co mpounds around degraded haustoria (Remus-Borel, 2005). Rodrigues et al. (2004) reported that Si stimulated the accumulation of momolactone phytoalexins in rice plan ts inoculated with M grisea Moreover, Ghanmi et al. (2004) detected accumulation of phenolic compounds around collapse d haustoria of powdery mildew infected epidermal cells of Arabidopsis thaliana. Fauteux et al. (2006) re ported that infection of Arabidopsis plants with powdery mildew fungi enhanced the expression of several defenserelated and metabolic genes and the addition of Si produced a more efficient defense response by the plant. However, they stated that Si al one had no effect on the metabolism of unstressed plants. In conclusion, alternative products have been effective in powdery mildew control of various crops. They have been as effective as fungicides when applied al one or in combination with other products. Therefore, it is worthwhile to evaluate al ternative methods for control of powdery mildew of gerberas in that products ot her than synthetic fungicides may help growers reduce their dependency on chemicals and consec utively, reduce the development of resistance by the pathogens. Also, the use of non-synthe tic fungicides may give growers an economic advantage by providing a potential market for environmentally fr iendly gerbera production.
37 CHAPTER 3 EVALUATION OF SILICON FOR SUPPRE SSING POWDERY MILDEW DEVELOPMENT IN GERBERA DAISIES Introduction Gerbera daisy ( Gerbera jamesonii Bolus ex. Hook f.) is an ornamental plant produced as a cut flower, potted plant and for landscape use (T ija and Black, 2003). In Florida, gerberas are mainly produced under greenhouse or shade house conditions as potted and bedding plants. Gerberas are susceptible to a va riety of pests and diseases. Po wdery mildew is an important fungal disease in gerberas and it can be caused by two species, Erysiphe cichoracearum DC.or Podosphaera (Syn. Sphaerotheca) fusca (Fr.) S. Blumer (Daughtrey et al., 1995). This disease affects all plant parts and reductions in yield and in quality are the most important components of economic loss. Environmental conditions that are most conductive for powdery mildew development include high humidity (80% to 90%) and moderate temperatures 20 to 28C (Daughtrey et al., 1995). These conditions are prevalent in Florida. Methods for powdery mildew management are chemical, cultural, and bi ological control. The majority of the nurseries in Florida use fungicides for powde ry mildew control such as ch lorothalonil, copper, mancozeb and metalaxyl (Larson and Nesheim, 2000). The de mand for high plant quality forces growers to intensify their spray programs against diseases which may lead to faster development of fungicide resistance while increasing overall prod uction costs. However, consumers awareness of the implications of pesticides (i.e. fungi cides) in environment and human health has intensified the search for altern ative methods of disease contro l (Gullino et al, 1999). Several alternative products have been reported to be eff ective for the control of powdery mildew such as electrolyzed oxidizing water in gerbera daisy (Mueller et al., 2 003b); clay in English cucumber and wine grapes (Ehret et al., 2001); phosphate and potassium salts in apple, cucumber, grapevines, mango, nectarine and wheat (Reuveni et al., 1996; Mitche ll and Walters, 2004;
38 Reuveni et al., 1998; and Reuveni and Reuveni, 1995); sodium bicarbona te in roses (Horst et al., 1992); salicylic acid, soy lecithin and sulfur, in chicory (Trdan et al., 2004); oils in cherry, cucurbits, grapevines and roses (Grove et al., 2005; Pasini et al., 1997; McGrath and Shishkoff, 2000; and Grove et al., 2000), cows milk in zu cchini squash (Bettiol, 1999) and biocontrol agents in rose and cucurbits (Pasini et al ., 1997; Romero et al., 2004). Another potential alternative for the control of pow dery mildew of gerberas is th e use of silicon, which has given satisfactory results in the ma nagement of powdery mildew of different crops such as Arabidopsis thaliana (Ghanmi et al., 2004); cucumber (Liang et al., 2005; Adatia and Besford 1986); cucumber, muskmelon, and zucchini squash (Menzies et al., 1992); grapes (Bowen et al., 1992); strawberry (Kanto et al., 2006) and wheat (Bela nger et al., 2003). Addition of silicon to the soil or the nutrient solution has show n beneficial effects on plants unde r biotic and abiotic stress (Ma et al., 2001). Among dicotyledonous plants, extensive resear ch has been conducted in greenhouse cucumbers where applications of silicon have been shown to reduce disease levels of powdery mildew caused by Sphaerotheca fuliginea (Schlecht Fr. Poll) also known as Sphaerotheca fusca (Fr.) S. Blumer (Epstein, 1999). Foliar and root applicati ons of potassium silicate reduced the number of co lonies of powdery mildew fungi in cucumber, muskmelon and zucchini squash (Menzies et al., 1992). Colony number of Uncinula necator in grape leaves was reduced to 11% of the control leaves when folia r silicon sprays were used (Bowen et al., 1992). Moreover, the effect of silicon has also been test ed in other components of parasitic fitness, and was found that increasing silicon amendments from 0.5 to 2.3 mM decreased colony number, colony area and percentage of c onidial germination (Menzies et al., 1991a). Powdery mildew development in Arabidopsis thaliana was limited and rarely observed when plants were watered with a nutrient solution containing soluble pota ssium silicate (Ghanmi et al., 2004). In Japan,
39 liquid potassium silicate was effective in suppres sing powdery mildew in the highly susceptible Toyonoka strawberry cultivar, when used in soil as well as in hydroponic cultivation (Kanto et al., 2004; Kanto et al., 2006). Bela nger et al. (2003) found that silicon amendments to the soil mix or added to the nutrient solution protec ted wheat plant from powde ry mildew caused by the fungus Blumeria graminis f.sp. tritic. The use of silicon in gerber a daisies was reported previous ly by Savvas et al. (2002) and Ritcher (2001). However, the objecti ve of their investigation was to evaluate the effect of silicon on yield and flower quality and on vase life, resp ectively. To our knowledge, the effect of silicon for the control of powdery mild ew in gerbera daisy has not be en investigated. Based on the effectiveness of silicon in controlling powde ry mildew in other crops, a hypothesis was formulated that silicon amendment to gerbera pl ants could decrease disease severity caused by Podosphaera fusca. Consequently, the objective of this st udy was to evaluate the efficacy of silicon for the management of powdery mildew in gerbera daisies grown under greenhouse conditions in Florida. Materials and Methods Experiments were conducted under greenhous e conditions from May 2006 to January 2007 at the University of Florida, Gulf Coast Rese arch and Education Center, Wimauma, Florida. Effect of Calcium Silicate in Powdery Mildew Development in Gerbera Plants Calcium silicate (CaSiO3) Slag 43.41% SiO2 (Calcium Silicates Corporation, Lake Harbor, FL) at rates of 0, 0.9, 1.8,3.6, 5.53 and 7.3 g/pot was added to the growing media CMA mix: peat moss 65%, perlite coarse 20%, A3 Coarse 15%, and rock 3% (Verlite Company, Tampa, FL). Afterwards, Osmocote Plus (15-9-12) cont rolled release fertilizer (The Scotts Company, Marysville,OH) at 11g/pot was added and all mixed with a concrete mixer (Gilson mixer 59015C, CF Gilco, Inc. Grafton, WI .). Gerbera seedlings Snow Wh ite Sunburst series (Twyford
40 International, Apopka, FL) highly susceptible to powdery mildew were transplanted into 15 cm diameter plastic pots previously filled with the growing medium 8 May 2006. Pots were placed on greenhouse benches and watered by drip irrigation. Powdery mildew caused by Podosphaera ( Sphaerotheca) fusca developed on plants from natural inoc ulum and symptoms were first seen two weeks after transplanting. Di sease evaluations were made at 7-day intervals beginning on 24 May and ending on 19 July 2006. The disease severity was rated using a 0 to 5 scale, where 0= no powdery mildew symptoms present, 1= 1 to 20%, 2= 21 to 40%, 3= 41 to 60%, 4= 61 to 80 % and 5= 81 to 100% of upper leaf surface covered with powdery mildew. This experiment was conducted as a randomized complete block design with six treatments and four replications. Disease ratings were used to calculate the ar ea under disease progress curve (AUDPC) for each treatment by the midpoint rule method (Campbell and Madden, 1990) as follows: AUDPC = i=1 n-1 [(yi + yi+1)/2] (ti+1 ti) where n is number of disease assessment times, y is the disease severity and t is the time duration of the epidemic. The AUDPC values obtained were analyzed by linear regression (SAS in stitute, Cary, NC). Effects of Silicon in Horticultu ral Traits of Gerbera Flowers Gerbera plants started blooming four week s after being transplanted. Flowers were harvested once they were fully developed, i.e. wh en the second circle of disks in the flower showed pollen development (Rogers and Tjia, 1990). Flowers were counted and the flower diameter, stem diameter, and flower height were recorded. Data were analyzed by linear regression (SAS institute, Cary, NC). Evaluation of Silicon Accumulation over Time This experiment was designed to determin e a timeline for silicon uptake into gerbera leaves. In August 2006, gerbera seed lings Snow White Sunburst series (Twyford International, Apopka, Florida) were transplanted into 15-cm di am plastic pots filled with the growing media
41 as previously described. Silicon was applied as Calcium silicate (CaSiO3) slag 43.41% SiO2 (Calcium Silicates Corporation Lake Harbor, FL) at 5.4 g/pot or as potassium silicate (K2SiO4) 26.5% SiO2 supplied as Kasil (PQ Corporation, Valley Forge, PA) at 0.27 mL/L (1.22 mM SiO2). Calcium silicate was incorporated into th e growing medium before transplanting and potassium silicate was applied thr ee times a week as a drench. Cont rol plants received no silicon. All plants were watered by drip irrigation. Each treatment had 45 plants th at were evaluated for silicon content on 2, 5, 9, 16, and 23 days after tran splanting (DAT). On each sampling date, four to five new leaves were collected from 9 plants of each treatment to determine silicon uptake. This experiment was repeated in January 2007. Data were subjected to an alysis of covariance using SAS Version 9.0 (SAS Institute, Cary, NC). Disease evaluations were made on 9, 16 and 23 DAT. The disease severity was rated using a 0 to 5 scale, as described previously. This experiment was conducted as a randomized comp lete block design with three treatments and three replications. Disease rati ngs were used to calculate th e area under disease progress curve (AUDPC) for each treatment as described pr eviously. The AUDPC values obtained were analyzed by analysis of variance (ANOVA) and m eans separated by the Wa llerDuncan k ratio t test (P 0.05). Silicon Extraction from Gerbera Leaves Four to five new leaves from gerbera plan ts were oven dried at 70C for 3 days. Dried tissue was ground finely with a sample mill (Cyclotec 1093, Foss analytical, Denmark) to pass through a 0.425 mm. Silicon content was determined by a modification of the autoclave-induced digestion procedure of Elliot and Snyder ( 1991). Briefly, 100 mg of dried, ground leaf tissue were placed in 100-mL polyethylene tubes and added 2 mL of 50% hydrogen peroxide (H2O2) and 3 mL of 50% sodium hydroxide (NaOH). Each tube was vortex gently and covered with loose fitting plastic caps. The tube s were placed in an autoclave at 103 kPa (15 psi) for 30 min. If
42 tissue was not completely digested, 2 mL of hydrogen peroxide were added to each tube, vortexed and placed in the autoclave again. Othe rwise, tubes were removed and the contents brought to 50 mL with distilled water. Silicon was determined colorimetrically as follows: a 1mL aliquot was taken from the dige sted plant tissue and mixed in 10 mL of distilled water. Then, 0.25 mL hydrochloric acid (1:1), 0.5 mL of ammonium molybda te solution (100g/L, pH 7.0), 0.5 mL tartaric acid (200g/L) and 0.7 mL of a reduc ing solution were added. The reducing solution was prepared by dissolving 4 g sodium sulfite (Na2SO3), 0.8 g 1-amino-2-naphthol-4-sulphonic acid, and 50 g sodium bisulfite (NaHSO3) in 500 mL water. Five minutes elapsed between the addition of the ammonium molybdate and the tartaric acid. A series of st andard silicon (silicon reference solution, Fisher Scientif ic) contents were developed and used to generate a regression equation for determining final silicon content in leaves (ppm). After 10 min, the absorbance was measured at 650 nm with a spectrophotomet er (PC 910, Brinkmann Instruments, Inc.). Results Effect of Silicon on Powdery Mild ew Development in Gerbera Plants Powdery mildew developed from natural inoc ulum and the first symptoms were observed 12 DAT. 26 DAT, disease severity was about 50% in all treatments. Disease increased until the end of the experiment when powdery mildew sy mptoms covered most of the leaves. Disease severity was not related to the amount of silicon applied at any of the ev aluation dates (Table 31). At the end of this experiment, leaves were collected to determine the silicon concentration and linear regression analysis showed no relationshi p of the silicon content in the leaves of the different silicon treatments to the amount applied (Table 3-2). Effects of Silicon in Horticultu ral Traits of Gerbera Flowers Gerbera flowers were evaluated for the effect of silicon on flower number, height, flower diameter and stem diameter. There was an average of 3 flowers per plant w ith an average height
43 of 21 cm, flower diameter of 7 cm and stem diameter of 0.5cm. There were no significant relationship of the amount of s ilicon applied and the average numb er of flowers, flower height, flower diameter and stem diameter (Table 3-3). Evaluation of Silicon Accumulation 2, 5, 9, 16 and 23 Days after Transplant Plants were treated with two sources of silicon, calcium silicate and potassium silicate, to evaluate silicon uptake over time (2, 5, 9, 16 and 23 days after transplant-DAT). This experiment was conducted twice and since the interaction betw een treatment and experiment was significant (P 0.05), results are presented separately. Fo r the first experiment, ANOVA showed no significant difference among the treatments applie d and nor was there a significant interaction between treatment and DAT. However, the DAT was significant s howing that silicon accumulation decreased over time as determin ed by the regression equation: Si (%) = 0.0550.00104(DAT). For the second experiment, silicon treatment (potassium silicate) had a significant effect, as did the interaction of potassium silicate and DAT (Table 3-4). The accumulation of potassium silicate in gerbera leav es showed a slightly increase over time as described by the regression equation: Si (%) = 0.037+0.00036(DAT). According to this equation, the expected Si concentration of gerbera leaves at 2 DAT woul d be 0.038 % of dry weight and it would increase to 0.045% at 20 DAT. Consequen tly, although the accumulation of potassium silicon in gerbera leaves is si gnificant, the amount of silicon did not vary greatly over time Powdery mildew severity was evaluated for the la st three weeks of this experiment to see if silicon uptake was related to disease control. Although plants treated with potassium silicate showed a slightly higher silicon uptake when comp ared to untreated control (Table 3-5), at the end of the experiment, the potassium silicate treatment did not significantly reduce disease (Tables 3-6, 3-7).
44 Discussion Silicon mixed with the soil or applied as a drench to gerbera plants did not reduce the severity of powdery mildew nor did it improve th e quality of the flowers. Disease symptoms in plants were observed 12 to 18 DAT and the sy mptoms were uniform among all treatments. The lack of control of powde ry mildew with silicon can possibly be explained by three hypotheses. First, the concentration of inocul um may be too high at the beginning of the experiment and there was not enough time for silic on to act before the fungus infected the plants. This is assuming that silicon moved systemically from the roots throughout the plant to either induce the plants defense mechanisms or accumu lated in plant tissues to create a physical barrier to the pathogen. In the wheat-powdery mildew pathosystem, calcium silicate was responsible for the accumulation of phenolic compounds that produced abnormalities in haustoria and thus reduced infec tion (Belanger et al., 2003). Liang et al. (2005) reported that Si applied to the roots of cucumber plants enhanced the production of pathogenesis-related proteins (PRs) in response to infection by Podosphaera xantii Silicon also induced the production of an electron-dense fungitoxic substance that appeared to be toxic to powdery mildew fungi infecting Arabidopsis thaliana (Ghanmi et al., 2004). Silicon has th e potential to trigger the plants defense mechanisms (Cherif et al., 1994; Ghanmi et al., 2004). Nevertheless, the exact time between uptake of silicon, m ovement throughout the plant an d the production of defense response is not clear. In some cases, plants are supplied with a silicon treatment a week or two before inoculation. Ma, et al. (2004) characterized the silicon upt ake in rice in a time-course experiment where roots and xylem sap were anal yzed for silicon. Silicon in the roots increased over time with an increase in the silicon c oncentration in the exte rnal solution, reaching saturation of about 5.5 mM after 25 h. The c oncentration of silicon in the xylem sap was measured after 8 h of silicon exposure and again, the silicon concentration in the xylem increased
45 with increasing of silicon in the external soluti on, but it was saturated at a higher concentration of about 35mM. This is in agreement with a similar study in cucumber where silicon uptake increased with time depending upon the silicon supp lied in the external so lution (Liang et al., 2005). Hence, in rice and in cucumber, the uptake of silicon can be a rapid active process and can provide good disease control. This apparently is not the case for gerberas especially since silicon did not seem to have any effect in supp ressing the development of powdery mildew. Silicon has been proposed to act as a physic al barrier in leaf surfaces for providing protection against biotic stress (Ma et al., 2001). Adatia and Besford (1986) found major effects of silicon in cucumber leaves as they had a rough er texture were more erect with a darker color and their senescence was delayed. Bowen et al. (1992) studied the effect of root and leaf applications of soluble silicon in powdery mildew severity of grapes. Reduction of disease severity was attributed in part to a physical barrier created by thick potassium silicate deposit in the leaf that limited hyphal penetra tion compared with areas of leaf surface that were not coated and fungal development was more extensive. However, they also considered the role of silicon in inducing a resistance response becau se of the movement of silic on and its deposition at fungal penetration sites. Kim at al. (2002) investigat ed the location of silic on in rice leaves using electron microscopy and X-ray microanalysis and found that silicon was deposited in epidermal cell walls, middle lamella and interc ellular spaces of ri ce leaves providing a physical barrier for the penetration and invasion of the pathogen Magnaporthe grisea, the cause of rice blast. Because of the deposition of silicon in plant cell walls, it is reasona ble to expect that silicon may strengthen plant tissues and thus creates a physical barrier that impedes direct penetration by fungal pathogens. Savvas et al. (2002) stated that silicon improved gerbera flower quality by providing mechanical strength to th e stems since their diameter increased with increasing silicon
46 concentration in the nutrient so lution. However, in our study, stem diameter was not affected by silicon. A second theory was that environmental condit ions such as temperature and light might have inhibited silicon uptake. Some experiments were conducted during the summer and plants were placed in a shady area of the greenhouse in an effort to lower temperatures and provide better conditions for the development of pow dery mildew. Schuerger and Hammer (2003) conducted an experiment to understand the discre pancy of powdery mildew control by silicon between studies conducted in Florida and Canada. Consequently, three horticultural parameters (cultivar, nutrient solution and rooting medium) and two environm ental factors (light intensity and temperature) were tested to determine th eir influence in silicon effectiveness to reduce powdery mildew severity in cucumber. Among al l factors tested, temperature had the most significant effect in reducing powdery mildew se verity. Temperatures of 20 C and silicon at 100 mg/L was demonstrated to be the most eff ective combination in decreasing the number of powdery mildew colonies per leaf compared to temperatures of 25 or 30C. These results could be expected based on the inter action between temperature, tran spiration and silicon uptake. Transport of water and solutes in the plants is regulated by active and passive processes (Ting, 1982) and transpiration plays a ro le in those transpor t processes (Taiz an d Zeiger, 1991). The process of uptake and movement of silicon in the plant is in the form of the uncharged molecule, H4SiO4, and its translocation within the plant is affected more by the tran spiration stream than with any other element (Epstein, 1999). Therefor e, silicon uptake can be influenced by the plants transpiration rate (Ma and Takahashi, 2002). Okuda and Takahashi (1962) demonstrated that silicon uptake in rice and tomato was influenced by transpir ation. When transpiration rate was reduced by high humidity, the silicon uptake in rice did not change, but it was reduced in
47 tomatoes. Transpiration is also highly influenced by humidity, te mperature, light and wind (Ting, 1982). Transpiration and photosynthesis in potato plants decreased when temperature increased above 25C (Ku et al., 1977). Light stimulates transpiration by opening the stomata hence increased transpiration might enhance Si uptake (Ma and Takahashi, 2002). The last theory is that gerbera plants do not uptake silicon. Savvas et al. (2002) reported that the inclusion of silicon in the nutrient solution improved flower quality by increasing the proportion of class I flowers and the thickness of fl ower stems. The authors stated that silicon also enhanced the uptake of calcium by the plants Silicon content in plant tissue varies greatly with the plant species and ex ternal silicon concen tration (Ma et al., 2001, Liang et al., 2006). Even genotypes within a species may have a diffe rent ability to uptake and accumulate silicon in their tissues (Epstein, 1994; Ma and Takahashi, 2002; Hods on et al., 2005). Comis (2007) reported that ornamentals such as New Guin ea impatiens, marigold and zinnia accumulate silicon as X-ray analysis showed significant concentrations on the leaves. Moreover, silicon reduced powdery mildew severity in Zinnia wh ile no effect was found in begonia and geranium as they did not accumulate silicon. Because of this specificity in silicon uptake and accumulation, testing different species and cultiv ars to determine their capacity to accumulate silicon is worthwhile. The amount of silicon in leaf tissues found in gerbera daisies, Snow-White Sunburst series, supplied with different rates of calcium silicate averaged 0.05% and did not increase with higher silicon rates. This result is different than t hose reported for rose, sunflower or rice. With rose and sunflowers, an increase in the amount of silicon applied to the soil or the nutrient solution resulted in increased silicon uptake from 0.02 to 0.09% and 0.5 to 3% respectively (Gillman et al., 2003; Liang et al., 2 006). Similarly in rice, Seebold et al. (2000) demonstrated that an increase in silicon supply produced an increase in silicon content of the
48 leaves. Calcium silicate at 500 kg/ha resulted in a silicon content of 3.1% and at double the rate, the concentration in the leaves increased to 3.5%. In our study, the same applied rates resulted in a silicon concentration in the le aves of 0.05% of dry weight and were not significantly different than the untreated control. So, while we have documented that gerbera daisy do not accumulate Si, the mechanisms of exclusion need to be established. Experiments on silicon uptake over time were conducted to determine a time course for silicon uptake in gerbera leaves. Silicon content in leaf or shoot tissues for 735 plant species was recently reported (Hodson et al., 2005). However, the time required for those plants to uptake silicon was not reported. Tamai and Ma (2003), in their characterization of silicon uptake by rice roots, determined that the uptake of silicon incr eased linearly with time and it took only hours for silicon to be absorbed. This is in agreement w ith Liang et al.(2006) who showed that for maize, rice, sunflower and wax gourd, it only took a few hours (2 to 10) for silicon to accumulate and the amount absorbed increased with an increase in silicon supply. Since silicon alone does not have fungicidal activity (Bowen et al., 1992), it must be absorbed before the pathogens attack to provide protection against dise ases and reduce disease severity. Usually, silicon treatmen ts start at the time when seed lings are transplanted and then plants are inoculated hours (Menzies et al., 1992) or weeks (Gillman et al., 2003; Rodrigues et al., 2001) later. Disease sympto ms are then observed within hours (Datnoff and Rutherford, 2004) or days (Gillman et al., 2003; Rodrigues et al., 2001; Menzies et al ., 1992) suggesting that, in most cases, silicon uptake and silicon response occurs in a matter of hours or days. However, this was not the case in our study with gerbera da isies. Plants treated with potassium silicate showed some evidence of silicon uptake 14 DAT and, at that time, symptoms of powdery mildew became apparent. However, no disease re duction was observed thereafter suggesting that
49 even though the plants had some silicon accumulation, it was not enough to reduce disease. Alternatively, the timing of obser vation may have been too short to make such conclusions and perhaps a longer time course expe riment would have shown greater silicon uptake, higher levels of disease and therefore differences in disease control. However, the first experiment to determine the effect of silicon on powdery mildew development showed that there were no significant differences in silicon c oncentration in the leaves of tr eated plants compared with the untreated control after two months. In our study on silicon content in ge rbera leaves receiving calcium silicate and potassium silicate, the silico n content ranged from 0.01% to 0.06% in a dry matter basis. This concentration is very low comp ared with silicon concentrations from 0.5% to 6% found in cucumber, strawberry, sunflower or rice tissues (Kanto et al., 2006; Liang et al., 2006; Seebold et al., 2000; Menzies et al., 1991b). In those experiments, silicon was successful in reducing disease severity. In the study with gerberas by Savvas et al. (2002), the authors never quantified silicon accumulation nor clearly stated th at the positive results in flower quality could be due to an indirect effect of silicon and not to its own propert ies. Therefore, the low silicon content in gerbera leaves may explain the lack of powdery mildew control. In conclusion, gerbera plants apparently do not accumulate silicon as fast and at high amounts as rice, cucumber or sunflower and, therefore, does not have an application for powdery mildew management under greenhouse conditions in Florida.
50Table 3-1. Effect of amending potti ng soil with calcium silicate (CaSiO3) on powdery mildew of gerbera daisy. Disease severity (0-5) z Days after transplanting CaSiO3 g/pot 12 19 26 33 40 47 54 61 68 AUDPCy 0 0.8 1.7 2.2 2.9 3.2 3.9 3.9 4.2 3.9 130.73 0.9 1.0 2.2 2.4 2.8 3.3 3.8 3.8 3.9 3.9 118.95 1.8 0.7 1.8 2.1 2.5 3.0 3.7 3.8 3.9 3.9 109.75 3.6 1.1 2.3 2.6 3.0 3.1 3.8 3.9 3.7 3.8 122.38 5.5 0.8 1.9 2.3 2.8 3.0 3.9 4.0 3.9 4.0 115.58 7.3 0.9 1.9 2.3 2.8 3.3 3.9 3.9 3.9 3.9 117.5 nsx ns ns ns ns ns ns ns ns ns zDisease severity rated on a 0 to 5 scale, where 0= no powdery m ildew symptoms and 5 = 81 to 100% of upper leaf surface covered with powdery mildew symptoms. yAUDPC = area under the disease progress curve xns: not significant
51 Table 32. Silicon (Si) content in new leaves of gerbera daisy. Siz (g/pot) % Si in leaves 0 0.049 0.9 0.046 1.8 0.079 3.6 0.041 5.5 0.048 7.3 0.037 nsy zCalcium silicate y ns: not significant Table 3-3. Effect of amending potti ng soil with calcium silicate (CaSiO3) in horticultural traits of gerbera flowers. Si (g/pot) Flower number No /plant Flower Height (cm) Flower diameter (cm) Stem diameter (cm) 0 3.27 19.81 7.13 0.54 0.9 3.21 19.89 7.08 0.53 1.8 3.63 21.46 7.27 0.54 3.6 3.33 22.53 7.81 0.56 5.5 3.23 21.54 7.45 0.53 7.3 3.67 22.16 7.14 0.55 nsz ns ns ns zns: not significant
52Table 3-4. Analysis of covariance for si licon content in gerbera l eaves at 2, 5, 9, 16, and 23 days after transplant (DAT). Exp 1 Exp 2 Source DF Mean Square F value Pr < F DFMean Square F value Pr < F Silicon treatment 2 0.00017337 1.05 0.352 2 0.00037337 4.49 0.013z DAT 1 0.00992459 60.25 <0.0001z1 0.00003427 0.41 0.5221 Treat*DAT 2 0.00004737 0.29 0.7505 2 0.00034887 4.19 0.0172z Error 0.00016472 0.00008317 z Significant different. Table 3-5. Silicon content in ge rbera leaves at 2, 5, 9, 16, and 23 days after transplant (DAT) Silicon treatment 0 Calcium silicate Potassium silicate DAT exp 1 exp 2 exp 1 exp 2 exp 1 exp 2 2 0.035 0.030 0.040 0.021 0.042 0.031 5 0.052 0.025 0.046 0.028 0.051 0.048 9 0.048 0.039 0.048 0.037 0.060 0.036 16 0.027 0.022 0.027 0.027 0.037 0.044 23 0.018 0.024 0.024 0.029 0.027 0.043 R2 exp. 1 0.36 R2 exp. 2 0.33
53 Table 3-6. Effect of silicon on powdery mildew severity in gerbera leaves. Experiment I Days after transplanting Si treatment 18 25 32 AUDPC 0 0.6 bz 1.0 a 1.8 a 15.37 a Calcium silicate 0.7 a 1.0 a 2.0 a 16.73 a Potassium silicate 0.6 ab 0.9 a 1.6 a 14.03 a zMeans in columns followed by the same letter are not significantly different according to the Waller-Duncan k ratio t test (P 0.05). Table 3-7. Effect of silicon on powdery mildew severity in gerbera leaves. Experiment II Days after transplanting Si treatment 18 25 32 AUDPC 0 0.2 az 0.3 ab 0.3 b 2.95 a Calcium silicate 0.1 ab 0.3 a 0.5 a 3.30 a Potassium silicate 0.1 b 0.2 b 0.5 a 3.00 a zMeans in columns followed by the same letter ar e not significantly different according to the Waller-Duncan k ratio t test (P 0.05).
54 CHAPTER 4 EVALUATION OF BIOFUNGICIDE PRODUC TS FOR MANAGING POWDERY MILDEW IN GERBERA DAISY Gerbera daisy ( Gerbera jamesonii Bolus ex. Hook f.) is an ornamental plant grown as cut flowers, potted plants and for landscape use (T ija and Black, 2003). In Florida, gerbera daisies are mainly produced under greenhouse or shad e house conditions as potted and bedding plants. Gerbera is susceptible to a vari ety of pests and diseases. Powdery mildew is an important fungal disease in gerbera and ca n be caused by two species, Erysiphe cichoracearum DC.and Podosphaera (Syn. Sphaerotheca ) fusca (Fr.) S. Blumer (Daughtrey et al., 1995). This disease may affect all plant parts and reductions in yield and in quality are the most important components of economic loss. Environmental co nditions most conducive for powdery mildew development include high relative humidity ( 80% to 90%) and moderate temperature (20 to 28C) (Daughtrey et al., 1995); these conditions that are common in Florida. The two main methods for powdery mildew control are repeated applications of fungi cides and the use of resistant or tolerant cultivars. Fungicides used in Florida for powdery mildew control include chlorothalonil (Bravo), Mancozeb + Thiophanate Methyl (Duosan ) and Propiconazole (Banner) (Larson and Nesheim, 2000). However, chemical control is not always completely effective since pathogens may develop resistance to so me fungicides (Gullino and Wardlow, 1999). In addition, consumer awareness of the implications of pesticides in the environment and human health has intensified the search for alternative methods of dis ease control (Gullino et al, 1999). Biofungicides are naturally base d microbial or biochemical products derived from animals, bacteria, plants, or minerals. These products can affect fungal organisms directly or may stimulate the defense response of the plant. They are generally narrow-spectrum, have low toxicity to non target organisms, decompose quickly, and thus are considered to have low
55 potential for negative impacts on the envir onment (McGrath, 2004; EPA, 2007). Biofungicides such as biological control agents (i.e. Bacillus subtilis ), potassium bicarbonate, phosphorous acid, Prevam and oils, are labeled for control of powdery mildew in ornamentals in Florida (Crop Data Management Systems, 2007). However, limited information is available on the effectiveness of these products in managing powdery mildew in ornamentals and more specifically on gerbera daisies. Consequently, th e objective of this study was to evaluate the efficacy of biofungicides for the management of powdery mildew in ge rbera daisy grown under greenhouse conditions in Florida. Materials and Methods Experiments were conducted under greenhouse conditions from April to June 2007 at the University of Florida, Gulf Coast Research and Education Center, Wimauma, Florida. Effect of Biofungicides in Powdery M ildew Development in Gerbera Plants: The growing medium (CMA mix: peat moss 65%, perl ite coarse 20%, A3 Coar se 15%, and rock 3%. Verlite Company, Tampa, FL) plus OP (Osmocot e Plus (15-9-12) contro lled release fertilizer (The Scotts Company, Marysvil le, OH) at 11g/pot, were combin ed using a concrete mixer (Gilson mixer 59015C, CF Gilco, Inc. Grafton, W I). Two highly susceptible (Snow White and Orange) and two moderately su sceptible (Hot Pink and Fuchs ia) Sunburst series (Twyford International, Apopka, FL) gerbera seedlings, were transplanted into 15-cm diam. plastic pots previously filled with the growing medium on 11 April, 2007. Sulfur (52% ) at the 6.2 ml/l rate (Micro Flo Company, Memphis, TN) was sprayed unt il runoff, on the leaves of all plants to eliminate any powdery mildew inoculum already present. Seven days after transplant (DAT), gerbera plants were sprayed with Actigard, Prevam, Milstop, K-phite AgSil (plus Tween20), Rhapsody (plus Biotune), Heritage alternated with Eagle as standard control or water (Table 41). Pots were placed on greenhouse benches and hand watered three times per week. Treatments
56 and cultivars were randomized and divided into two benches (96 plants per bench). Treatments were applied weekly on the upper surface of the l eaves to runoff with a hand sprayer. Powdery mildew, caused by Podosphaera ( Sphaerotheca ) fusca developed on the plants from natural inoculum 28 DAT. Disease evaluations were made at seven day intervals beginning 9 April (28 DAT)and ending 15 June(65 DAT) 2007. Disease severity was rated on a 0 to 6 scale, where 0= no powdery mildew symptoms, 1= 1 to 20%, 2= 21 to 40%, 3= 41 to 60%, 4= 61 to 80 %, 5= 81 to 99% and 6= 100% of upper leaf surface cove red with powdery mildew symptoms. This experiment was conducted as a completely randomi zed design with 8 treatments and 4 cultivars. Disease severity ratings were analyzed by week by analysis of variance (ANOVA) with mean separation by Fisher's Protected LSD (P 0.05) (Statistix 8.1, Tallahassee, FL). Disease ratings were used to calculate the area under disease progress curve (AUDPC) for each treatment by the midpoint rule method (Campbell and Madden, 1990) as follows: AUDPC= i n-1[(yi + yi+1)/2] (ti+1 ti) where n = the number of disease assessment times, y = disease severity and t = time duration of the epidemic. AUDPC values were transformed to square roots to normalize variance and then s ubjected to analysis of variance followed by Fisher's Protected LSD (P 0.05) (Statistix 8.1, Tallahassee, FL) to separate means. Results Powdery mildew developed from natural inoc ulum. Disease severity was assessed once a week for six weeks. There were significant differences (P 0.05) between treatments and cultivars. The interaction between treatment and cultivar was significant (P 0.05). However, the F value for the interaction was low (F=3.11) thus disease severity ratings of treatments and cultivars were pooled for comparisons among treatments and cultivars (Tables 4-2, 4-3).
57 Until 40 DAT, the average relative humidity was below 80% and temperature fluctuated from 72 to 76 F (22-24 C). At about 44 (DAT), the relative humidity increased to above 80% (Figure 4-1).On 44 DAT, powdery mildew increas ed on all cultivars and then it gradually progressed every week thereafter (Figure 4-2). The first symptoms were observed 28 DAT, but incidence was very low and during the first two weeks of evaluations (28 to 37 DAT), disease was observed only on untreated plants and those treated with Agsil and Actigard. Most of the plants at 44 DAT were infected except for plants that received the Heritage-Eagle treatme nt. A week later, disease increased for all treatments including the Heritage-Eagle-treated plan ts (Table 4-2). During the last three weeks of the experiment (51 to 65 DAT), disease severity increased gradua lly regardless of treatment or cultivar (Tables 4-2, 4-3). By the end of the expe riment, non-treated plants had a disease severity of 4.2. Plants treated with Actigard and AgSil ha d a disease severity of 2.1 and 1.7, respectively. Rhapsody-treated plants had a disease severity of 1.0 whereas less than 0.6 was observed on plants treated with Heritage-E agle, K-phite, Milstop, and Prevam (Table 4-2). As indicated by the AUDPC values, all treatments we re significantly different from untreated control. However, none of the biofungicide treatments were as effective as the commercial fungicides. Nevertheless, K-phite, Milstop, Prevam, and Rhapsody reduced di sease severity from 0.3 to 1.0, approximately between 76 to 93%, compared with untreated plan ts (Table 4-2). Regardless of treatment, cultivar Snow White and Orange were the most susceptible to powdery mildew throughout the experiment. Cultivars Hot Pink and Fuchsia were not significantly different from each other when ev aluated on a weekly basis. However, the AUDPC values showed that Fuchsia was the least sus ceptible among all cultivars tested (Table 4-3).
58 Heritage alternated with Eagle significantly reduced disease severity on all cultivars when compared with untreated contro l (Figure 4-3). For the cultivar Fuchsia, Milstop and Prevam significantly reduced powdery mild ew severity to a level equivalent to that of commercial fungicides. Moreover, Prevam was as effective as the standard fungicides in reducing disease severity in more susceptible cultivars such as Hot Pink and Orange (Figure 4-3). K-phite and Milstop were the only biofungicide products capa ble of reducing powdery mildew on the most susceptible cultivar, Snow White. Moreover, K-phite was as effective as th e rotation of fungicide Heritage and Eagle. Discussion Disease symptoms appeared almost a month after transplant and the powdery mildew epidemic developed slowly thereafter. During the first six weeks of the experiment, the relative humidity was below 80% and since powdery mildew develops best at a high humidity (80 to 90%) (Daughtrey et al., 1995), the low relative humidity probably was a constrain to a faster epidemic development. However, adverse microc limatic conditions were probably useful for the plants cells that were already attacked by powdery mildew fungi in that they reduced the speed of infection process giving the plan t more time to transport material to the infection site and stop penetration by formation of papilla e (Aust and Hoyningen-Huene, 1986). The biofungicides products Actigard, Agsil, K-phite, Milstop, Pr evam, and Rhapsody suppressed powdery mildew of ge rbera daisies compared with unt reated plants under greenhouse conditions in Florida. However, these products were not as effective as th e fungicide program of Heritage alternated with Eagle. Among all biofungicides, Actigard and Agsil were the least effective treatments. Rhapsody provided moderate disease control and K-phite, M illstop and Prevam were the most effective in reducing powdery mildew severity. Moreover, for th e cultivar Fuchsia, Milstop and Prevam were
59 as effective as the fungicide program. For the most susceptible cultiv ar, Snow White, K-phite and Milstop were the only biofungicide products capable of reducing powdery mildew severity. Actigard did not provide satisfactory control of powdery m ildew in gerbera daisies when compared to the other biofungicide treatment s. Similarly, Babadoost (2002) reported that Actigard did not provide a satisfactory level of powdery mildew control in pumpkin. In addition; this material was ineffective when used agains t powdery mildew of cucumber, suggesting that it failed to enhance plant defense responses (Wur ms et al., 1999). However, when Actigard was used in muskmelon, it provided moderate powdery mildew control when compared to other treatments (Matheron and Porc has, 2003). When used in cabbage and lettuce, Actigard was effective in controlling powdery mildew (Matheron and Porchas, 2004; Miller and Hernandez, 2001). Thus, these results are in agreement with t hose of Oostendorp et al (2001) and Grlach et al. (1996) who suggested that the crop may determ ine the activation of resistance. Consequently, Actigard may not elicit a strong defe nse response in gerbera plants. Powdery mildew severity on gerbera daisies treated with AgSil was significantly different that untreated plants, although the level of disease reduction obtained was low when compared with other biofungicide treatments. However, pot assium silicate has been demonstrated to be effective in suppressing powdery mildew in othe r crops (Belanger et al ., 2003; Bowen et al., 1992; Ghanmi et al., 2004; Kanto et al., 2006; Me nzies et al., 1992). Furthermore, potassium silicate was effective in suppressing powdery mildew on the highly susceptible strawberry cultivar, Toyonoka, in soil as well as in hydropo nic cultivation (Kanto et al., 2006, 2004). The lack of satisfactory powdery mildew suppression by potassium silicate on gerbera daisies was demonstrated previously in chapter 3. In the previous experiment, drench treatment with potassium silicate was ineffective but plants were exposed for only 32 days. In the present
60 experiment, plants were exposed for 56-days and foliar treatment with pota ssium silicate still did not provide the disease reduction reported for ot her crops (Belanger et al., 2003; Bowen et al., 1992; Ghanmi et al., 2004; Kanto et al., 2006; Menzies et al., 1992). Given that Bowen et al. (1992), Kanto et al. (2007) and Menzies et al. (1992) in their in vitro assays demonstrated that conidial germin ation and germ tube elongation of powdery mildew fungi was unaffected by potassium s ilicate, suppression of powdery mildew by potassium silicate may be due to its ability to induce systemic acquired resistance. Subsequently, the lack of effective response of AgSil might be in agreement with the lack of response of Actigard and ultimately, the plant species may determine the activation of resistance (Grlach et al., 1996; Oostendorp et al., 2001). In a previous study, Rhapsody failed to control powdery mildew of gerbera and a high level of disease severity wa s observed when compared to other treatments (Sconyers and Hausbeck, 2004). However, Bacillus spp., provided 80% reduction in powdery mildew severity of cucumber, caused by P. fusca, as determined by in vitro studies on detached leaves and seedlings (Romero et al., 2004). In this st udy, gerbera plants treated with Rhapsody had significantly lower disease sever ity than untreated plants; howe ver, the effect was moderate compare to other biofungicide treatments. This is in agreement with Utkhede and Koch (2006) who showed that Bacillus subtilis (Quadra 137), significantly redu ced powdery mildew severity in cucumber when compared with untreated pl ants although other non chemical products tested provided better results than B. subtilis The efficacy of biological control varies with the level of relative humidity prevalent in the gree nhouse (Belanger et al., 1994). However, Bacillus strains are resistant to adverse environmental condi tions (Shoda, 2000). Regardless, in our study, Biotune was added to Bacillus subtilis to enhance its efficacy and reduce the dependency of high
61 humidity. Therefore, the moderate response of Rhapsody for control of powdery mildew of gerberas may be due to causes other than low humidity in the greenhouse. In this study, K-phite was effective in reduci ng powdery mildew of gerbera compared with untreated plants and other biof ungicide products. However, it did not provide the same level of control as the commercial fungici des. Similar results were reporte d previously by Mueller et al. (2003a) who showed that phosphorous acid applie d as Biophos was effective for control of powdery mildew of gerbera daisies as wa s Fosphite on powdery mildew on muskmelon (Matheron and Porchas, 2005). Schi lder et al. (2003) reported that ProPhyt reduced powdery mildew incidence and severity on grapes as much as the fungicide program (Dithane/Abound/Ziram). Our results did not corres pond with those of Schilder et al. (2003) in that K-phite was not as effective as the comm ercial fungicide products for powdery mildew control. Consequently, the fungici de program used in their study was not as effective as the one used in this study or phosphite products acted differently for each pathosystem. Our study demonstrated that pota ssium bicarbonate formulated as Milstop reduced powdery mildew levels in gerber a daisies and, for cultivar Fuchsi a, the level of disease reduction was comparable to that of the systemic fungici des. Sconyers and Hausbeck (2004) showed that gerbera plants Jaguar Mix trea ted with Milstop had low levels of infection similar to those found in plants treated with Heritage and Ea gle. Furthermore, Uchida and Kadooka (2001) showed that Kaligreen reduced le vels of powdery mildew to le ss than 5% in gerbera plants grown in Hawaii. Potassium bicar bonate products have also been successful in reducing powdery mildew on other crops such as cucumber, musk melon, pumpkin, roses, sweet peppers, tomato and winter squash (Matheron and Porchas, 2003; McGrath and Shishko, 1999; Dik et al., 2003).
62 Among the biofungicide products evaluate d for gerbera powdery mildew, Prevam was the most effective. Moreover, the level of disease reduction for cultivars Fuchsia, Hot pink and Orange compared to that achieved with the system ic fungicides. Prevam was previously reported to provide effective control of powdery mildew of strawberries (Merte ly et al., 2005). Based on its components (boron; 0.99%, orange oil and orga nic surfactants; 99.01%), it is possible the oil ingredient in Prevam breaks down fungal mycelia and spores and exposes them to desiccation and thus prevents further infection. Oil has b een used to control diseases for many years (Calpouzos, 1966) and it has been effective in reducing powdery mildew of apple, cherry, cucurbits, grapes and roses. The level of disease reduction ranged from highly effective (McGrath and Shishkoff, 1999, 2000; Pasini et al., 1997) to slight ly satisfactory (Fernandez et al., 2006; Grove et al., 2005). In some cases, th e efficacy of oils to reduce powdery mildew, compared to the levels obtained with standa rd fungicides (Northover and Schneider, 1996), were even superior (Grove et al., 2000; Wojdyla, 2002). All cultivars performed as expected; Snow wh ite and Orange were the most susceptible and Fuchsia and Hot pink were less susceptible. Our study is the first evaluation of severa l biofungicide products for the control of powdery mildew of gerberas under greenhouse conditions in Florida. In addition, this is the first study demonstrating that Prevam significantly re duced powdery mildew severity in gerbera daisies. Alternative products such as Rhap sody, Milstop, Kaligreen, Biophos and electrolyzed oxidizing water were previously reported for cont rol of powdery mildew of gerberas in other states including Georgia, Hawa ii and Michigan (Mueller et al ., 2003a,b; Sconyers and Hausbeck, 2004; Uchida and Kadooka, 2001).
63 In conclusion, the biofungicide products tested when applied prior to disease infection may reduce powdery mildew significantly compared to no treatment. As a consequence these products can be used as part of an integrated disease management program as an alte rnative to reduce the use of standard fungicides for the c ontrol of powdery mildew in gerbera daisy.
64 Table 4-1. Source, rate, active ingredient, and manufacturer of biofungici des used to suppress powdery mildew in gerbera daisy. Product name Rate /liter Active ingredient Manufacturer Heritage alt. 0.3 g azoxystrobin 50% Syngenta, Greensboro, NC Eagle 0.4 g myclobutanil 40% Dow AgroSciences. Indianapolis, IN Prevam 4 ml sodium tetraborohydrate decahydrate 0.99% Oro Agri, Inc. Trophy Club, TX AgSil 21 + Tween 20 3 ml + 100 l potassium silicate (12.65%K2O, 26.5%SiO2 / polyoxyethylene polyoxyethylene (20) sorbitan monolaurate PQ Corporation. Valley Forge, PA./ Fisher Scientific Inc. K-phite 5 ml monoand dipotassium salts of phosphorous acid 53% Plant Food Systems, Inc. Zellwood, FL Milstop 3 g potassium bicarbonate 85% BioWorks, Inc. Fairport, NY Actigard 0.1 g acibenzolar-S-methyl 50% Syngenta, Greensboro, NC Rhapsody + Biotune 10 ml + 1.3 ml Bacillus subtilis (QST 713) 1.34% + Adjuvant AgraQuest, Inc. Davis, CA. / AgraQuest, Inc. Davis, CA
65 Table 4-2. Effect of biofungici des and conventional fungicides on powdery mildew severity in gerbera daisies. Days after transplantzy Treatment 28 37 44 51 58 65 AUDPCyx Control 0.1 ab 0.3 a 0.8 a 1.6 a 3.1 a 4.2 a 7.31 a Heritage alt. Eagle 0.0 c 0.0 c 0.0 d 0.1 e 0.1 e 0.2 e 1.21 f Prevam 0.0 c 0.0 c 0.1 d 0.2 de 0.4 de 0.3 de 2.23 e Agsil + Tween 20 0.0 bc 0.0 bc 0.3b 0.7 bc 1.1 b 1.7 b 4.52 b K-phite 0.0 c 0.0 bc 0.1 cd 0.4 dc 0.6 cd 0.6 d 3.13 cd Milstop 0.0 c 0.0 c 0.0 d 0.3 de 0.3cde 0.6 cd 2.58 de Actigard 0.1 a 0.1 ab 0.3 bc 0.8 b 1.4 b 2.1 b 4.87 b Rhapsody + Biotune 0.0 c 0.0 c 0.1 bcd 0.5 cd 0.7 c 1.0 c 3.50 c zDisease severity rated on a 0 to 6 scale, wher e 0= no powdery mildew symptoms, 1= 1 to 20%, 2= 21 to 40%, 3= 41 to 60%, 4= 61 to 80 %, 5= 81 to 100% and 6= 100% of upper leaf surface covered with powdery mildew symptoms. yMean separations in columns follow by the same letter are not significantly according to Fishers protected LSD (P 0.05). xArea under disease progress curve (AUDPC) values.
66 Table 4-3. Effect of treatments on powdery mild ew severity in gerbera cultivars treated with biofungicides and conven tional fungicides. Days after transplantingzy Cultivar 28 37 44 51 58 65 AUDPCyx Snow white 0.1 a 0.1 a 0.3 a 0.8 a 1.2 a 1.5 a 4.12 a Hot pink 0.0b 0.1 ab 0.3 a 0.5 bc 0.8 b 1.1 b 3.58 b Fuchsia 0.0 b 0.0 b 0.1 b 0.4 c 0.6 b 1.0 b 2.96 c Orange 0.0 b 0.1 ab 0.2 a 0.6 ab 1.2 a 1.7 a 4.01 a zDisease severity rated on a 0 to 6 scale, wher e 0= no powdery mildew symptoms present, 1= 1 to 20%, 2= 21 to 40%, 3= 41 to 60%, 4= 61 to 80 %, 5= 81 to 100% and 6= 100% of upper leaf surface covered with powdery mildew symptoms. yMean separations in columns follow by the sa me letter are not significantly according to Fishers protected LSD (P 0.05). xArea under disease progress curve (AUDPC) values.
67 Figure 4-1. Daily average temper ature and relative humidity in greenhouse from April to June, 2007. Figure 4-2. Disease progression of powdery mildew for the ge rbera cultivars used in the biofungicide experiment.
68 Figure 4-3. Area under disease prog ress curve (AUDPC) values for severity of powdery mildew in gerbera daisies treated with biofungicide s and conventional fungicides. Bars with the same letter in each cultivar do not differ significantly according to Fishers protected LSD (P 0.05).
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83 BIOGRAPHICAL SKETCH Catalina was borne in Medellin, Colombia. She earned a B.S. degree in agronomy from Zamorano UniversityHonduras, in 1999. Catalina came to the U.S. in 2001 to work as an intern at the hydrology department with the Forest Service and the Bu reau of Land Management in Roseburg, Oregon. There, she met her husband, Sco tt Moyer, who she married in 2003. A year later, they moved to Florida. Catalina worked at Dr. Natalia Peres stra wberry pathology lab for ten months before she became a graduate student. In 2007, Catalina finished her graduate program and received a M.S degree in plant path ology. Catalina will continue working with Dr. Peres at GCREC in Wimauma, FL.