Determining the infection process of Phoma macrostoma that leads to bioherbicidal activity on broadleaved weeds

Biological Control 59 (2011) 268–276

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Determining the infection process of Phoma macrostoma that leads to bioherbicidal activity on broadleaved weeds K.L. Bailey a,⇑, W.M. Pitt b, F. Leggett c, C. Sheedy c, J. Derby a a

Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada S7N 0X2 National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia c Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 b

a r t i c l e

i n f o

Article history: Received 31 March 2011 Accepted 30 June 2011 Available online 7 July 2011 Keywords: Bioherbicide Dandelion Taraxacum officinale Fungus Phoma macrostoma Infection process Histopathology Macrocidins

a b s t r a c t Isolates of the fungus Phoma macrostoma cause intense photobleaching and mortality of Canada thistle (Cirsium arvense), dandelion (Taraxacum officinale) and other broadleaved weeds when applied to the soil. The symptoms are caused by the production of macrocidins which have been extracted from cultured mycelium and the growth medium broth. The objective of this study was to determine the pathway of infection that leads to plant damage resulting in bioherbicidal activity. This was accomplished by first determining which infective unit (i.e., conidia and/or mycelium) resulted in plant damage when applied to a host. Then the infection process was microscopically observed from infested granules placed in the soil through the various stages of colonization and penetration in a resistant and a susceptible host. Conidia were ineffective as infective units because there was no plant damage when target weeds were inoculated using either a foliar spray of a conidial suspension or granules containing conidia placed in the soil. Only mycelium of the fungus applied either pre-emergently to soil ahead of weed seed emergence or post-emergently to soil containing established weeds resulted in significant plant damage. Microscopic observations showed that P. macrostoma 94-44B mycelium germinated from formulated granules in soil, colonizing roots of dandelion (susceptible) and barley (resistant) within 7 days of application. The fungus entered the hosts at sites proximal to root hairs where wounding of the cells was most likely, and growing intercellularly towards the root core. In dandelion, the mycelium proliferated around the vascular trachea disrupting the competence of neighboring cells. In barley, proliferation was less obvious being restricted to the outer layers and there was no disruption of the internal cell structure. P. macrostoma boasts potential as a bioherbicide to control susceptible broadleaved weeds but not harm resistant nontarget hosts. Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.

1. Introduction Phoma macrostoma Montagne is a ubiquitous, plurivorous fungus that occurs on a wide range of hosts including more than a dozen members of the Rosaceae (Boerema and Dorenbosch, 1970). Although the fungus is frequently characterized as a weak or opportunistic parasite, invading hosts via wounds or lesions created by other pathogens (Boerema and Dorenbosch, 1970; Boerema et al., 2004), it has a cosmopolitan distribution and occurs not only on a wide variety of woody trees and shrubs (Boerema and Dorenbosch, 1970), but also on grasses, pasture legumes (Johnston, 1981), artichoke (Kubota and Abiko, 2002), and raspberry (Williamson and Hargreaves, 1979).

⇑ Corresponding author. Fax: +1 306 956 7247. E-mail addresses: [email protected] (K.L. Bailey), [email protected] (W.M. Pitt), [email protected] (F. Leggett), [email protected] (C. Sheedy), [email protected] (J. Derby).

Under natural field conditions, P. macrostoma survives as mycelium in living hosts or leaf litter (Boerema et al., 2004; Bengtsson et al., 2006), reproducing asexually via the formation of pycnidia from which conidia are released and disseminated in a salmon to flesh-colored matrix, usually by way of rain-splash or occasionally by wind (Rai, 1985; Boerema et al., 2004). The primary function of conidia is for distance dispersal whereas mycelial growth is slow and restricted to soil and in plants. Conidial germination on hosts has not been studied in the literature, but on agar we observe conidia to germinate within 24 h forming long germ tubes that create a mycelial mat. To date, the sexual stage of P. macrostoma has not been observed. A number of strains of P. macrostoma were isolated from necrotic lesions on Canada thistle (Cirsium arvense (L.) Scop.) plants collected from fields and roadside ditches throughout the Canadian provinces of Alberta, Saskatchewan, Ontario, New Brunswick, and Nova Scotia (Zhou et al., 2004). When mycelial fragments of these isolates were applied to soil containing broadleaved weed species, such as dandelion (Taraxacum officinale Weber ex F.H. Wigg.), chlo-

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K.L. Bailey et al. / Biological Control 59 (2011) 268–276

rosis and bleaching of leaf tissues on a continuous spectrum from green through yellow to white, henceforth known as ‘photobleaching’, occurred and death ensued, yet neither of these symptoms were observed when monocotyledonous weeds and crop species were exposed in the same way (Bailey and Derby, 2001). Of 14 strains of P. macrostoma collected that exhibited bioherbicidal activity, all were genetically similar (Zhou et al., 2005). One isolate, P. macrostoma 94-44B has been earmarked for development as a potential bioherbicide, specifically targeting broadleaved weed control in turfgrass. For this type of application, the fungus is grown on grain, producing mycelium, which is then formulated into a granule for broadcast dispersal to the soil. However, while mycelium is the predominant infective unit, the role of conidia was uncertain. Hence, the objectives of this study were to verify the capacities of conidia and mycelium to act as the infective units of this potential bioherbicide that lead to plant damage and death, and microscopically pursue the bioherbicidal infective unit through the infection process from an infested granule in the soil to colonization of a resistant and susceptible host. 2. Materials and methods 2.1. Determination of infective units resulting in bioherbicidal activity Three experiments were designed to study the morphology of the fungal inoculum and application methods of the bioherbicide on plant damage. These experiments were conducted over a period of several years in order to address emerging questions on the mode of action of this fungus as a potential bioherbicide. As a result, several different hosts and three fungal isolates (94-44B, 9554A1, and 97-12B) were used, with each isolate demonstrating bioherbicidal activity. The fungal cultures have been deposited as IDAC230201-3, IDAC230201-5, and IDAC230201-6, respectively, at the International Depositary Authority of Canada, Winnipeg, MB. Cultures were maintained by cryopreservation of hyphal fragments and conidia in a 1:1 skim milk (10% w/v) to glycerol (40% v/ v) solution stored at 80 °C. Isolates were revived by thawing a vial to room temperature and spreading the contents on 9 cm diameter Petri plates containing 15 ml potato dextrose agar (PDA) augmented with 3 ml l 1 of 85% lactic acid (Zhou et al., 2004). 2.1.1. Experiment 1 Experiment 1 evaluated the bioherbicidal activity from an application of conidia of P. macrostoma isolates 95-54A1 and 9712B to emerged shoots of Canada thistle plants using a post-emergent foliar spray, and of mycelium of these fungi to emerging shoots using a mycelial-agar mat applied pre-emergently to the soil. Fungal isolates for both treatments were cultured in 10-cm2 Petrie plates containing full strength potato dextrose agar (PDA; Difco Laboratories, Detroit, Michigan, USA) at 23 ± 5 °C on a laboratory bench in a room with a 16 h photoperiod comprising a mixture of natural daylight from windows and overhead fluorescent light in the room. After 14 days, plates were flooded with sterile distilled water and conidia gently dislodged from the mycelial agar-mat surface by scraping with a sterile glass rod. Subsequent conidial suspensions obtained via this method were pooled then diluted to a concentration of 1  106 conidia/ml with sterile distilled water. Using an air brush sprayer (20–30 psi), 4 ml aliquots of the diluted conidial suspension were applied to 1–3 shoots of Canada thistle plants at the ‘bolting stage’ of development (stalk elongation and flowering). Canada thistle plants were grown from cloned root stock in field soil for at least 21 days prior to inoculation, and subsequently cultivated in 10-cm2 pots for the purpose of the study. Control plants were inoculated with sterile distilled

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water using the same technique. Inoculated and control plants were then placed in a Percival dew chamber (Percival, Boone, Iowa, Model E54DLV) at 20 ± 2 °C at 98–100% relative humidity for 48 h, with 24 h darkness followed by 8 h light (T5 bulbs with a 4100 color spectrum, 22 lmol m 2 s 1), and a further 16 h of darkness. After 48 h, plants were removed from the dew chamber and placed in a temperature-controlled glasshouse with a diurnal light regime comprising a 16 h photoperiod provided by artificial light from high-pressure sodium lamps (230 lmol m 2 s 1). Glasshouse temperatures ranged from a daytime high of 20 ± 5 °C to an overnight low of 15 ± 5 °C and pots were watered daily to runoff using a misting nozzle. Petrie plates containing fungal cultures washed free of conidia were then used in a mycelial agar-mat bioassay. Four pieces of 10-cm long Canada thistle root taken from the same clone were placed on the soil surface of 10-cm2 plastic pots filled to 3=4 capacity with soil-less planting mix (one part sand to 12 parts of a 1:2 sphagnum peat moss: vermiculite mixture amended with 1% of 16:8:12 (N:P2O5:K2O) and 0.2% of 0:20:0 (N:P:K) controlled-release fertilizers on a weight per volume basis (The Scott’s Company, Marysville, Ohio). The contents of the Petrie plates were then extracted and inverted such that the mycelial surface lay flat on the soil in direct contact with the roots. A small quantity of soil-less mix was then applied as a thin cover layer over the contents of each pot to prevent moisture loss and disturbance during watering. Petrie plates containing fresh, uncultivated PDA were used as controls. Pots were transferred to the temperature-controlled glasshouse comprising the diurnal light, temperature and watering regimes described previously. Bioherbicidal activity was measured 3 weeks after inoculation. All pots were rated for the degree of photobleaching based on a scale of 1–6, with 1 representing healthy green shoots and 6 having white necrotic shoots. Foliar fresh weights (g) were taken by clipping the plants off at the crown-soil line. Canada thistle leaves do not have high moisture content, so dry weight analysis was not performed. Data on fresh weights were subjected to analysis of variance (ANOVA) and the fungal isolates were compared to their respective controls using a Dunnett’s test (P = 0.05). The means and ranges of the photobleaching ratings using the interval scale were presented. The experiment was conducted twice using a completely randomized block design with five replications. 2.1.2. Experiment 2 Experiment 2 evaluated the bioherbicidal activity of a range of conidial and mycelial formulations of P. macrostoma isolate 9444B on stands of dandelion and a turfgrass mixture 21 days after treatment. Five 0.5-cm diameter plugs were extracted from a 14day old culture of 94-44B grown to the edge of the plate on full strength PDA and used to inoculate each of six 500 ml Erlenmeyer flasks containing 125 ml of potato dextrose broth (PDB; Difco Laboratories). Flasks were sealed aseptically with aluminum foil, placed on an orbital shaker oscillating at 150 rpm, and cultivated for 30 days at ambient temperatures (23 °C) with a 16 h photoperiod provided by natural and fluorescent light. After 30 days incubation, the contents of all six flasks were pooled, and using nylon mesh, the solid mycelial fraction was separated from the broth and conidial portion by filtration and then washed with sterile distilled water to remove remaining broth. A ‘cell free’ broth was then prepared from the liquid fraction by passing 150 ml of the broth containing conidia through a 0.45-lm cellulose acetate filter. Conidia retained on the filter surface were retrieved and collected by reverse filtration using sterile distilled water. The solid fraction was weighed and 15 g wet weight of mycelium added to 100 ml of sterile distilled water and homogenized using a Polytron PT10-35 homogenizer (Kinematica, Luzern, Switzerland). From the resulting suspensions, six treatments, out-

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lined below, were prepared for application either via foliar spray or soil drench; (1) conidia in sterile water applied as a foliar spray (1  107 conidia/ml), (2) conidia in broth applied as a foliar spray (1  107 conidia/ml), (3) cell-free broth (devoid of conidia and mycelium) applied as a foliar spray, (4) 15% w/v mycelial homogenate applied as a foliar spray, (5) 15% w/v mycelial homogenate applied as a soil drench (10-ml/pot), and (6) a sterile water control, also applied by spray to the foliage. The experiment was conducted using a completely randomized block design with the six treatments and three replications, with all experiments performed twice. To simulate pre-emergent and post-emergent bioherbicide applications, a sowing regime was devised such that two sets of treatment pots were produced. The first set of pots contained established turfgrass with a ready population of emerged dandelion at the time of treatment, while the second set contained only turfgrass to which dandelion seed was then sown at the time of treatment. Hence, 14 days prior to treatment, 0.11 g of turfgrass seed (‘Over Seeding’ mixture, Early’s Farm and Garden Centre, Saskatoon, SK) was sown into 10-cm2 pots filled to the 3=4 mark with soil-less planting mix. Half of the total number of pots were then broadcast with dandelion seed at a rate of 20 seeds/pot, and along with the rest of the pots, watered with a misting nozzle and placed in the temperature-controlled glasshouse. After 14 days, turfgrass stands were trimmed in all pots and in the pots created for the ‘post-emergent’ treatments, number of established dandelions recorded, such that a ‘before treatment dandelion count’ could be attained. The remaining pots earmarked for ‘pre-emergent’ treatment were sown with dandelion at a density of 20 seeds/pot, thereby rendering both application models ready for treatment. Foliar spray applications used a cabinet track sprayer (Research Instrument Company, Guelph, Ontario) with an XR8002 nozzle employing a pressure of 2.7  105 Pa at a height of 50 cm above the plant canopy and a crossover speed of 4.6 s. To verify conidia and mycelial viability following spray application, an agar plate containing PDA was placed in the spray cabinet alongside recipient plants and evaluated for fungal growth after 24 h incubation at 23 °C. Soil drench applications were completed by pouring 10-ml of the 15% w/v mycelial homogenate onto the surface of the pots, with the intention that the drench makes contact as best as possible with the entire soil surface. Treated pots, separately grouped by pre- or post-emergent application, were then placed in two Percival dew chambers (previously described) at 20 ± 2 °C and 98–100% relative humidity for 48 h, with 24 h darkness followed by 8 h light, and a further 16 h of darkness. Pots were then transferred to the temperature-controlled glasshouse for 21 days. Treatments were assessed by recording the total number of dandelions/pot and the number of photobleached dandelions/pot. A rating of >1 on the 1–6 photobleaching scale was considered to be photobleached. Percent photobleaching of dandelion was calculated by dividing the number of photobleached plants in the pot by the total number of plants in the pot and multiplying by 100. Dandelion leaves and grass leaves were clipped at the soil interface and fresh weights (g)/pot were taken. Unlike pre-emergent applications for which dandelion counts can only be based on the total number of dandelions present at the end of the experimental period regardless of health, the post-emergent applications used the final numbers of dandelion to be compared with ‘before treatment counts’. This method accounts for dandelions that may die throughout the experimental period and whose existence would otherwise go unnoticed. If assessments were conducted in ignorance of this, percent photobleaching scores would be based only on the number of individuals remaining at the succession of the experiment, often undervaluing the post-emergent result. Data were subjected to an ANOVA and means comparison using Duncan’s Multiple Range Test (P = 0.05).

2.1.3. Experiment 3 Experiment 3 examined the bioherbicidal activity of foliar spray and soil granular applications of mycelium and conidia of P. macrostoma isolate 94-44B on purple loosestrife (Lythrum salicaria L.) and dandelion 21 days after treatment. Purple loosestrife seeds were collected on May 20, 2008 from dried seed heads of naturally-occurring plants originating in Saskatoon, SK. Dandelion seed was obtained from Richter’s Seed, Goodwood, ON. Five weeks prior to treatment, both purple loosestrife and dandelion plants were established in the glasshouse. Five seeds/pot placed at equidistant points were sown on the surface of 10 cm2 pots filled to the 3=4 mark with soil-less planting mix, and covered with a further 1mm layer of soil. Pots were then placed in plastic trays and transferred to the temperature-controlled glasshouse, and watered daily with a misting nozzle. Six treatments, including two controls outlined below, were prepared using P. macrostoma 94-44B; (1) DIGS adjuvant mixture (0.1% dextrose, 1.5% Intac, 0.05% gelatin and 0.15% Silwet L-77) as a formulation control sprayed, (2) conidia in DIGS for foliar spray (1.0  107 conidia/pot), (3) mycelium in DIGS for foliar spray (1.0  107 colony forming units (cfu) of mycelial fragments/pot), (4) conidia in granules for soil broadcast (rate of 128 g/m2 containing 2.2  107 viable conidia/pot), (5) mycelium in granules for soil broadcast (rate of 128 g/m2 containing 1.3  104 cfu/pot), and (6) an untreated control. A granular formulation control was not included because previous work had shown no phytotoxicity (Bailey et al., 2010b). However, the foliar spray carrier DIGS did show some phytotoxicity in previous experiments (unpublished data), so a DIGS only control was deemed necessary to separate carrier effects from those of the bioherbicide. Conidial suspensions were made by flooding multiple 7– 10 day-old PDA plates containing P. macrostoma 94-44B, with 10 ml sterile distilled water, and dislodging the conidia with a sterile glass rod. Conidial suspensions were pooled such that a dilution series could be prepared, whereby 50 ml of suspension containing 2  106 conidia/ml in a DIGS carrier solution could be prepared. A mycelial homogenate was produced by cultivating P. macrostoma 94-44B in PDB under constant agitation (orbital shaker at 150 rpm) for 7–14 days at 23 ± 5 °C. Mycelium was collected by filtration through cheese cloth and 3 ml of drained mycelium homogenized for 20 s in 47 ml DIGS solution, such that the final homogenate contained 2.0  106 cfu/ml. Formulated granules containing mycelium for soil application were made by a proprietary process which involved growing the fungus on grain and then formulating using extrusion and spheronization technology producing granules with 1.3  106 cfu/g. Formulated granules containing conidia for soil application were made from 5 ml of a stock solution of conidia combined with 150 ml water and 375 g of barley flour yielding granules containing 1.7  107 conidia/g. Foliar spray applications were performed using an air brush sprayer (1.7  105 Pa), with plants being sprayed to run off (5 ml/pot). To verify conidia and mycelial fragment viability following spray application, three agar plates containing PDA supplemented with 0.3% lactic acid (PDA-La; stock conc. lactic acid 85%; EM Science, Merck KGaA, Darmstadt, Germany) were sprayed alongside recipient plants for each treatment and evaluated for fungal growth after 24 h incubation at 23 °C. Plants were then placed in a Percival dew chamber (previously described) at 20 ± 2 °C at 98–100% relative humidity for 48 h, with 24 h darkness followed by 8 h light, and a further 16 h of darkness before being moved to the temperature-controlled controlled glasshouse. Soil applications with the granular formulations were broadcast to the surface of the pots and then placed on benches in the same glasshouse. Plants were rated for photobleaching 7 and 14 days after treatment using the photobleaching rating scale described in Experi-

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ment 1. The means and ranges of the photobleaching ratings using the interval scale were presented. Due to uneven growth in the replicates of purple loosestrife, biomass data is only presented for dandelion. Biomass data were subjected to ANOVA and means comparison using Duncan’s Multiple Range Test (P = 0.05). The experiment was conducted twice using a completely randomized block design with five replications. 2.2. Microscopy study on root colonization by mycelium 2.2.1. Host inoculation and root preparation To document fungal interactions and host specificity of P. macrostoma 94-44B, root colonization studies were conducted on resistant (barley) and susceptible (dandelion) species. Dandelions were cultivated from seed, individually transplanted to 15.5-cm diameter plastic pots filled to 3=4 with soil-less planting mix, and nurtured for a period up to 8 months, before being broadcast with 128 g m 2 of P. macrostoma 94-44B granules prepared from cultured grain. Barley cv. ‘CDC Gainer’ was sown at the rate of 5 seeds/pot placed at equidistant points on the surface of 10-cm2 pots filled to the 3=4 mark with soil-less planting mix and covered with a further 1-mm layer of soil before being broadcast with 128 g m 2 of P. macrostoma granules 94-44B. Barley was known to be resistant so plant death would not occur after treatment thus permitting observations to be taken on younger plants. In contrast, dandelion was known to be highly susceptible with seedlings dying within a few days of exposure to the bioherbicide. Hence, more mature dandelion plants were inoculated. Untreated controls were prepared likewise, but broadcast with ground uncultured grain. Pots were then transferred to a temperature-controlled glasshouse. Roots were collected from plants 7 and 28 days after application. Roots were gently washed to remove large clumps of soil and blotted dry with sterile filter paper. Root tip sections (2–5 cm long) were removed and sealed in 20 ml scintillation vials containing FAA fixative (10 ml of 37% formaldehyde, 5 ml glacial acetic acid, 50 ml 95% ethanol, and 35 ml distilled water) for a minimum of 24 h. Ten replicate samples were taken at each time interval and the inoculation and root sampling procedures were conducted twice. 2.2.2. Staining and microscopy The whole roots were removed from FAA fixative and rinsed three times for 30 min in distilled water, dehydrated through a series of ethanol dilutions; 30%, 50%, 70%, 95%, 95%, absolute ethanol (containing eosin dye), at 1 h each. The samples were infiltrated through an absolute ethanol: xylene series (3:1, 1:1, 1:3) for 1 h each, then placed in pure xylene for two changes. The samples were put in an oven at 60 °C and paraffin chips were added to the xylene solution until saturated. Pure melted paraffin was gradually added to the samples until only pure wax remained and they were embedded in paraffin in preparation for sectioning. Root sectioning (25 lm) was performed on a Reichart-Jung Autocut rotary microtome with glass knives. Sections were arranged and adhered to glass slides on warming trays. Paraffin was removed by rinsing slides twice in xylene for 10–15 min, followed by rinsing twice in an absolute ethanol solution for 10 min each. Sections were then rehydrated for 10–15 min in a range of graded ethanol solutions diluted with water (95%, 70%, and 50%) before being stained. Sections were stained for 45 min in Pianese IIIB (0.01 g martius yellow, 0.5 g malachite green, 0.1 g acid fuchsin and 50 ml 95% ethanol in 150 ml distilled water), rinsed in absolute ethanol, stained for a further 2 min and rinsed with distilled water, before being decolorized in acidified 95% ethanol for approximately 30 s, washed with xylene, and mounted in permount. This staining procedure was used on whole roots and root sections. It resulted in differential staining that visually distinguished

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between fungal growth (stained red) and plant structures (stained green). Microscopic examinations were conducted on a Nikon Microphot-FXA microscope fitted with a Nikon digital camera. Digital images were recorded and observed using Image J and Adobe Photoshop. Some sections containing fungus were observed using the Olympus Fluoview FV 1000 Confocal Laser Scanning Biological microscope (Olympus Corporation, Tokyo, Japan). These images were viewed using the 488 and 543 nm laser lines with the dwell time of 20.08 ls with section depth ranging from 0.35 to 2.05 lm/ section depending on the sample. 2.2.3. Production of polyclonal antibodies and labeling of P. macrostoma In addition to staining, all microscopy samples were labeled with a P. macrostoma-specific polyclonal antiserum, thereby confirming that the presence of the fungal material in whole roots and root sections was in fact P. macrostoma 94-44B. To obtain antiserum specific against P. macrostoma 94-44B, two female New Zealand white rabbits were each immunized as follows; P. macrostoma mycelium was lyophilized and ground to a powder (250 lg) which was suspended in a solvent consisting of 250 ll of phosphate buffered saline (PBS, pH 7.4) and 250 ll of Titermax Gold adjuvant (Sigma–Aldrich). Immunogen (250 ll) was administered subcutaneously at two sites at the back of the neck every 14 days for a total of 6 weeks commencing on the first day of the study, henceforth known as day 0 (i.e., days 0, 14, 28, and 42). Blood was collected using the ear vein bleeding technique every 14 days commencing on day 7 of the study (i.e., days 7, 21, and 35). The ear vein was slightly pinched using a clamp, and blood retrieved directly from the ear vein using a syringe fitted with a 22-gauge needle. Blood was transferred to a 1.5 ml microfuge tube and processed to obtain serum (1 h at 37 °C, overnight at 4 °C). Blood serum was transferred to cryogenic vials and stored at 20 °C until used. Antibody titer and specificity were tested against P. macrostoma 94-44B by enzyme-linked immunosorbent assay (ELISA) and Western Blotting. Once a sufficiently high titer was reached, terminal cardiac bleed was performed. The antiserum with the highest affinity and titer was used on all microscopy samples of P. macrostoma 94-44B in order to confirm its presence. Samples that had been previously stained were destained by soaking the slides in xylene to remove the cover slip, after which the slides were rehydrated through a series of alcohols to water. Both fresh and destained samples were rinsed in double distilled water overnight and then placed in a citrate buffer (pH 6.2) and incubated in a scientific microwave three times each for 5 min at 95 °C using 100 W at power setting 1. Samples were rinsed in tris-buffered saline (TBS) with 1% bovine serum albumin (BSA) for 15 min, then placed in diluted antiserum (1/1000 ll for 1 h and rinsed again with TBS/1% BSA for 15 min. Samples were placed in dilute AlexaFluor 546 (1/1000 ll) for 1 h before rinsing in double distilled water for 15 min before being mounted in Mowiol medium (2.4 g Mowiol 4–88, 6 g glycerol, 6 mL distilled water, and 12 ml 0.2 M Tris pH 8.5) to reduce fading, and the sample was then covered or placed in a dark room to dry. Root samples were again observed with an Olympus Fluoview FV 1000 Confocal Laser Scanning Biological microscope. 3. Results 3.1. Determination of the bioherbicide infective units In Experiment 1, mycelium of P. macrostoma applied to the soil in advance of Canada thistle shoot emergence (pre-emergently) caused significant fresh weight reduction in plants when compared to untreated controls (Table 1). In contrast, conidia of P. macros-

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Table 1 The effect of applying conidia of Phoma macrostoma 95-54A1 and 97-12B postemergently to the foliage, or mycelium pre-emergently to the soil, on resulting foliar biomass (g) of Canada thistle. Data presented are the mean of two trials. Treatment

Conidia to foliage fresh wt. ga

Mycelium to soil fresh wt. ga

Untreated control 95-54A1 97-12B

4.4a

17.4a

5.1a 5.0a

0.2b 0.0b

a Different letters within a column indicate significant deviation from the control according to Dunnett’s test at P = 0.05.

toma applied post-emergently to the foliage of Canada thistle failed to reduce fresh weight. Similarly, the application of mycelium caused significant photobleaching with 95-54A1 and 97-12B scoring 5 and 6, respectively compared to the untreated control at 1 (with no range variation in the ratings). However, the application of conidia did not produce a high degree of photobleaching with 95-54A1 and 97-12B scoring 3 (range 2–3) and 1 (no range variation), respectively alongside the control at 2 (range 1–2). In Experiment 2, cultivation of P. macrostoma 94-44B in liquid culture produced photobleaching on dandelion and reduced significantly foliar fresh weight as a percentage of the water control (Table 2). While mycelial soil drench treatments produced the most photobleaching and greatest fresh weight reduction of dandelion, mycelial spray treatments behaved similarly, albeit slightly less effectively when applied pre-emergently. Likewise, both the ‘cellfree broth’ and ‘conidia in broth’ treatments incited significant photobleaching and foliar fresh weight reduction of dandelion. But conidia in water produced no photobleaching or biomass reductions in dandelion and had the same effect as the water-only control. None of the treatments caused photobleaching or fresh weight reduction in turfgrass. In Experiment 3, conidia and mycelium of the fungus were administered both to dandelion and purple loosestrife by way of foliar spray and granular formulation. With the application of a soil granular formulation containing mycelium, photobleaching on dandelion achieved a rating of 3 (no range variation) after 7 days and increased to 4 (no range variation) after 14 days, at which time symptoms also appeared on the growing tips of purple loosestrife

(mean = 2, range 1–2). Similarly, the foliar spray comprising homogenized mycelium also caused slight photobleaching on dandelion (mean = 2, range 1–3). However, none of the other treatments produced symptoms on either host. Most notably, formulations containing conidia of the fungus neither encapsulated within the soil granule nor combined with the DIGS carrier were able to incite a response in either host. The DIGS carrier itself also failed to invoke a response. Hence, the fresh weight of dandelion was reduced only by application to the soil of a mycelium-containing granule (Table 3). All other treatments failed to reduce fresh weight and were more similar to the untreated control.

3.2. Microscopy study of root colonization by mycelium Roots of dandelion and barley (P. macrostoma-susceptible and resistant hosts, respectively) were assessed microscopically for evidence of root colonization 7 and 28 days after treatment with mycelium-containing granules of P. macrostoma 94-44B. Dandelion treated with the fungus showed typical photobleaching symptoms when assessed at both 7 and 28 days after treatment. In contrast, barley displayed no symptoms of photobleaching at either interval. Fig. 1 traces the growth of P. macrostoma 94-44B (stained red) from the granule broadcast on the soil to attachment of the fungus on the dandelion root. Fungal mycelium was observed to be viable and proliferated from the grain-based granular formulations used as inoculum (Fig. 1a) growing towards and attaching to the surface of the dandelion root (Fig. 1b). Thereafter, root sections taken at 7 days showed the mycelium inside root hairs and congregating over cells proximal to and along the length of the outer epidermis (Fig. 1c). At a deeper layer, the mycelium was present in the central area of the root but appeared to be moving over and under the cells without penetrating them (Fig. 1d). The formation of appressoria and penetration pegs that would allow entry into the cells were never observed. Although mycelium never penetrated cells, it continued to proliferate intercellularly reaching the central core of the root (Fig. 1e), and then proceeded to breakdown the internal plant cellular structure by 28 days (Fig. 1f). A section taken just above the vascular trachea showed extensive proliferation of mycelium parallel to and around these vascular structures (Fig. 1g), but despite fungal presence on the exterior of the trachea, the interior portion was not colonized (Fig. 1h). In comparison, a section

Table 2 The effect of a range of conidia, mycelium and culture broth formulations of Phoma macrostoma 94-44B on dandelion photobleaching and foliar fresh weight (g), and turfgrass foliar fresh weight (g) when applied pre and post-emergently as a spray or soil drench. Data are the means of two trials. Treatment

Post-emergent application Water Control Conidia in water Conidia in broth Cell free broth Mycelial spray Mycelial soil drench Pooled standard error of mean Pre-emergent application Water Control Conidia in water Conidia in broth Cell free broth Mycelial spray Mycelial soil drench Pooled standard error of mean

%Photobleaching dandeliona,b

Foliar fresh weight (g) (% of control) Dandeliona

Turfgrassa

0.0a 5.2a 89.2b 95.2b 86.4b 95.0b 6.4

100.0a 86.7a 15.7b 15.9b 24.9b 26.4b 15.7

100.0a 97.3a 98.7a 106.6a 99.1a 100.4a 9.3

0.0a 0.0a 34.1b 59.3bc 72.9c 100.0d 15.1

100.0a 100.0a 58.6ab 12.4c 36.1bc 0.0c 33.2

100.0a 119.4a 114.8a 102.9a 131.2a 127.7a 20.7

a Different letters within a column indicate significant differences between treatments according to Duncan’s Multiple Range Test at P = 0.05. b % Photobleaching = the number of photobleached plants divided by the total number of plants in the pot multiplied by 100.

K.L. Bailey et al. / Biological Control 59 (2011) 268–276 Table 3 The effect of conidia and mycelium formulations of Phoma macrostoma 94–44B on dandelion foliar biomass. Data presented are the means of two trials. Host

Treatment

Foliar biomass g/pota

Dandelion

Untreated control DIGS only Foliar spray conidia + DIGS Foliar spray mycelium + DIGS Soil granule with conidia Soil granule with mycelium Pooled standard error of mean

54.2b 46.7b 68.8a 61.8ab 65.7ab 22.9c 3.8

a

Different letters within a column indicate significant differences between treatments according to Duncan’s Multiple Range Test at P = 0.05.

through an untreated dandelion root showed no evidence of fungal mycelium in or around the trachea (Fig. 1i). In barley plants exam-

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ined 7 days after treatment, fungal colonization was observed along the surface of the root which was positioned between two bioherbicide granules (Fig. 2a). However, even after 28 days, mycelium was restricted to the root hairs and the exterior outer root edges with no evidence of extensive proliferation, cell penetration, or cell disruption in the resistant host (Fig. 2b). 4. Discussion Irrespective of their status as weak, wound or opportunistic pathogens, Phoma species have previously been considered as candidates for biological control of invasive weeds including Canada thistle (Guske et al., 2004; Kluth et al., 2005), and dandelion (Neumann and Boland, 2002; Stewart-Wade and Boland, 2004; Schnick and Boland, 2004). Whereas conidia are usually considered the most infective of fungal propagules (Boyette et al., 1991), and

Fig. 1. Root colonization of dandelion (green) following inoculation with Phoma macrostoma 94-44B (red); (a) fungal mycelium proliferating from formulated granules, (b) growth and attachment of mycelium on dandelion root 7 days after treatment, (c) fungal mycelium associated with root hairs 7 days after treatment, (d) longitudinal section showing mycelial growth over and under interior root cell layers 7 days after treatment, (e) intercellular mycelial proliferation 28 days after treatment, (f) destruction of central core cell contents 28 days after treatment, (g) extensive mycelial proliferation parallel to vascular trachea 28 days after treatment, (h) mycelial growth around but not penetrating trachea 28 days after treatment, and (i) lack of mycelial presence around vascular trachea in untreated dandelion 28 days after treatment. Images shown were stained with Pianese IIIB, except 1b which was stained using the antibody.

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Fig. 2. Root colonization of barley following inoculation with Phoma macrostoma 94-44B; (a) fungal colonization on the epidermis of a barley root 7 days after treatment, and (b) mycelial growth in the root hair and longitudinal growth restricted to the outer root cell layers 28 days after treatment. Images shown were stained with Pianese IIIB.

therefore the most suitable form of the fungus for bioherbicide formulations, mycelia are often dismissed as a useful source of infective propagules because they are thought to be fragile and thus more prone to desiccation on the leaf surface (Lawrie et al., 2001). Whether by virtue of conidia or homogenized mycelium, infective propagules of candidate bioherbicides have, for the most part, been applied to the target weed via foliar application. Successful infection via this method requires that a number of important parameters be optimized, not least of which is the requirement of a period of free moisture or dew, for up to 12 h after application (Auld and Morin, 1995). In addition to this, research has shown that only around 5% of the volumes of aerial applications of herbicides ever reach the target weed (Combellack, 1981; Pimental and Levitan, 1986). Hence, the successful application of infective propagules, especially conidia, to the foliage of target plants, is a significant challenge. In this study, we tested a range of conidial and mycelial suspensions in order to establish methods for the delivery of infective and efficacious inoculum of P. macrostoma to dandelion and other target weeds. While foliar applications of homogenized mycelium, cell free broth, and broth containing conidia, produced weak symptoms on dandelion, only mycelial formulations either homogenized or granularized, and applied directly to the soil surface provided consistent and significant plant damage. Soil mycelial compositions caused a high level of photobleaching and effectively reduced the biomass of pre-emergent stands of Canada thistle and both pre- and postemergent dandelion. Both foliar and soil applications using conidia and mycelium were ineffective against purple loosestrife indicating that this weed would likely not be a target host. While previous studies have similarly utilized mycelium of a Phoma species as a source of inoculum against dandelion (Stewart-Wade and Boland, 2004), this is the first study to consider nonfoliar pre-emergent application of infective propagules for dandelion control. Our studies have shown that P. macrostoma causes damage to dandelion when mycelium is applied to the soil both pre- and post-emergently. If a single dandelion plant can produce from 2000 to 23,000 seeds/plant (Roberts, 1936; Bostock and Benton, 1979), a pre-emergent application for control of the dandelion seed bank would be beneficial in reducing the weed population. Some researchers suggest that seedlings are more susceptible to biological agents than mature plants (Boyette and Walker, 1985; Cardina et al., 1988), although others have found the opposite to be true

(Makowski, 1993). In studies utilizing Phoma herbarum Westend, dandelion seedlings were most susceptible to infection (Neumann and Boland, 2002), while 3-month old seedlings of salal succumbed to foliar applications of Phoma exigua Desm. within 14 days (Zhao and Shamoun, 2005). The latter treatments however only succeeded in defoliating mature plants, which subsequently re-grew from uninfected tissues (Zhao and Shamoun, 2006). P. macrostoma has a unique biological property that should also be considered when evaluating the infective units and the infection process. Bioherbicidal activity does not rely solely on fungal attachment, germination, and penetration of the host to initiate symptoms. Under certain conditions, P. macrostoma produces phytotoxic compounds within its mycelial mass and excess quantities may be exuded to the surrounding growth medium (Graupner et al., 2003). These phytotoxins, known as macrocidins, are produced solely by the mycelium of the fungus (and not the conidia) and cause growth retardation of shoots and roots along with severe photobleaching (Graupner et al., 2006). The formulation of P. macrostoma mycelium into granules and its application to the rhizosphere encourages both infection and uptake of these phloemmobile compounds by the plant roots. When considering the infection process, it is the combined role of infection and presence of macrocidins that have a direct effect on the plant itself. Some of the macrocidins will be released from the granule in the moist soil which is taken up by plant roots. At the same time, the fungus germinates from the granules and invades the host entering through natural openings associated with root hairs. Mycelium then grows towards the root core, breaking down the internal cellular structure of susceptible plants, while simultaneously producing additional phytotoxins that are directly absorbed by the vascular system, further accentuating the damage. Resistant plants are both asymptomatic to the presence of macrocidins and have restricted fungal growth within the root illustrating that susceptibility is dependent on the combined effects of at least two modes of action. To date, we are aware of few examples where fungi that produce phytotoxic compounds have been cultured on solid substrates and incorporated into the soil as a broad-spectrum bioherbicide (Jones et al., 1988; Heraux et al., 2005; Hutchinson, 1999). When grown on peat moss amended with sucrose and ammonium nitrate, the mycoparasitic fungus Gliocladium virens J.H. Mill., Giddens & A.A. Foster (syn. Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx produces a phytotoxin, viridiol. The

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phytotoxic activity of the fungus is substrate-dependent, and when colonized by fungal hyphae, air-dried, ground to a powder and applied to the soil, causes severe necrosis of germinating seed radicles and subsequent seedling death in a number of crop and weed species (Jones et al., 1988). However, without a high nutrient substrate, the fungus is unable to produce growth-suppressive levels of viridiol. Hence, T. virens is constrained by the availability of inexpensive substrates, as peat is largely a nonrenewable resource (Jones and Hancock, 1987). Recently, both Heraux et al. (2005) and Hutchinson (1999) reported that cultivation of T. virens on composted chicken manure supplemented with nutrients facilitated viridiol production at levels capable of controlling the emergence and growth of green foxtail (Setaria viridis (L.) P. Beauv.) and redroot pigweed (Amaranthus retroflexus L.). But this time, phytotoxicity to non-target crop species was a concern for field applications as none of the crops tested by Jones et al. (1988) were sufficiently tolerant to recommend direct sowing of crops into the fungus-peat mix. Jones et al. (1988) and Howell and Stipanovic (1984) have suggested that more localized applications of the mycoherbicide might be necessary to overcome such issues, yet despite these constraints commercial products such as SoilGard™ (formerly GlioGard™), containing T. virens as the active ingredient are utilized in the field (Ricard, 1981), albeit as mycofungicides to control soilborne fungi such as Rhizoctonia solani Kuhn., and Pythium ultimum Trow. Like T. virens, P. macrostoma produces phytotoxins, which are detrimental to plant growth and reproduction. Similarly, these phytotoxins are substrate-dependent (unpublished data). When formulated into a granule using extrusion and sphaeronization technology, the fungus remains viable during extended periods of storage and requires no special handling or re-hydration (Bailey et al., 2010a). Broadcast to the soil surface, granules infused with fungal mycelium provide both pre-emergent and post-emergent control of dandelion, and several other broadleaved weeds, but in contrast to T. virens, can be used with selected nontarget crop species. To be an effective bioherbicide, mycelium of P. macrostoma must be applied to the soil as a granule. Conidia are not capable of inducing photobleaching or biomass reductions. In both resistant and susceptible hosts, mycelium emergent from the granules colonized the surface of the root epidermis, entered the roots via the hairs, and grew intercellularly in the outer cell layers. In susceptible hosts, the foliar tissues became photobleached and the mycelium grew to the central vascular core of the roots, proliferated extensively around the vascular trachea and caused internal root cell disruption. In resistant hosts, there was no photobleaching and mycelial growth was less extensive and restricted to the outer epidermal edges. These visual observations provide evidence of two modes of action which affect the efficacy and therefore the susceptibility of target plants to P. macrostoma: that being the action of phytotoxic macrocidins and the capacity of the host to prevent extensive mycelial growth through root tissues. Acknowledgments The Scott’s Company and Agriculture and Agri-Food CanadaMatching Investment Initiative provided funding under a collaborative research and development agreement between industry and the Canadian federal government. The authors would like to thank Jennifer Sundstem and Michael Mendenhall for technical support. References Auld, B.A., Morin, L., 1995. Constraints in the development of bioherbicides. Weed Technology 9, 638–652. Bailey, K.L., Derby, J., 2001. Fungal isolates and biological control compositions for the control of weeds. US patent application Serial no. 60/294475. Filed May 20, 2001, pp. 73.

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