Fate and Management of Turfgrass Chemicals - American Chemical

Chapter 18

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In Vitro Screening and Recovery of Rhizoctonia solani Resistant Creeping Bentgrass: Evaluation of Three In Vitro Bioassays 1,3

2

M. Tomaso-Peterson , A. Sri Vanguri , and J. V. K r a n s 1

2

2

Departments of Entomology and Plant Pathology and Plant and Soil Sciences, Mississippi State University, Mississippi State, MS 39762

Over the past decade there has been increased environmental awareness of fungicide use on golf courses. A need has been identified within alternative pest management programs for rapid in vitro bioassays to screen and select turfgrasses with enhanced resistance to pathogenic fungi. The Host-Pathogen Interaction System (HPIS), fungal extracts, and direct fungal inoculation are in vitro bioassays evaluated for rapid screening and selection of Rhizoctonia solani resistance in creeping bentgrass. Penncross callus that survived cocultures with R. solani regenerated reduced numbers of plantlets. A reduction in tissue necrosis was observed in all in vitro bioassays when R. solani-selected plantlets were screened with R. solani isolates compared to non-selected plantlets.

Rhizoctonia blight of creeping bentgrass (Agrostis stoloniferous var. palustris Huds.), incited by Rhizoctonia solani Kiihn, has been a persistant turfgrass problem since the disease was first documented in the early 1920s (6). In cool season grasses such as creeping bentgrass, the disease is more prevalent during summer months. Initial symptoms in bentgrass turf appear as small rings (10-15 cm dia), bluish-gray in color. Necrotic lesions develop on leaf blades, and as they coalesce, blades become totally necrotic. As the disease progresses throughout the turf, brown patches up to 50 cm in diameter become evident. Rhizoctonia blight can be controlled by cultural practices including mowing height, fertility, and irrigation management, supplemented with an effective fungicide program. 'Penncross' creeping bentgrass set the standard as the premier putting green turf in thel950s. This is attributed to high density, dark color, aggressive growth habit, and uniformity in a putting green surface. However, Penncross is susceptible to R. solani. At least seven cultivars have been developed from Penncross using traditional breeding methods to improve turf density, color, and overall quality, while increasing tolerance to brown patch (26).

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Current address: Box 9655, Mississippi State, MS 39762.

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© 2000 American Chemical Society

Clark and Kenna; Fate and Management of Turfgrass Chemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


315 Years of research and funding are spent developing new turfgrass cultivars. Traditional breeding methods include selection, re-selection, plot tests, seed production, and finally turf evaluations at selected sites before a cultivar is commercially released. A preliminary step that can increase efficiency in selecting improved plant types is in vitro screening. The foundation of in vitro screening is based on somaclonal variation. Somaclonal variation accounts for the phenotypic and genotypic variations that occur when plants are cultured in vitro and has been exploited as a tool to recover improved plant types (72). This selection tool is used to eliminate cells that cannot withstand the selection pressure. Many horticultural and agronomically important crops have improved disease resistance via in vitro selection (1,9,19). In vitro selection for enhanced disease resistance may employ direct (fungal inoculum) or indirect (culture filtrates, fungal extracts, or purified toxins) methods for selection (7,13,15,18,22,25). The objective of this research was to develop and evaluate direct and indirect in vitro bioassays for rapid screening of Rhizoctonia blight resistance in creeping bentgrass. Materials and Methods The host plant used throughout the bioassays was 'Penncross' creeping bentgrass. Penncross callus was obtained according to procedures described by Krans et al. (11). Isolates of R. solani were maintained on potato dextrose agar (PDA) (39 g L" , Sigma, St. Louis, MO) in the dark at 25°C. 1

Host-Pathogen Interaction System (HPIS). The HPIS is an in vitro cell selection bioassay developed primarily for increasing the efficiency of recovering callus that can survive the presence of a pathogenic fungus. The HPIS consists of a double-sided, two compartment, Lutri-plate modified for use with callus and a pathogenic organism (28). Compartment A was adapted to meet the requirements for pathogen growth. A gas exchange port was made in the lid of compartment A by cutting a hole using a heated #3 cork borer. A sterile, 13-mm glass fiber disk was sealed over the hole using a nontoxic glue. This port facilitated movement of gases out of the compartment once sealed and prevented the entrance of contaminants. Thirty-seven milliliters of sterile water agar (20 g agar L" , Difco, Detroit, MI) were poured into compartment A and allowed to solidify. Sterile bentgrass leaf blades were placed on the water agar surface, serving as a natural substrate for the fungus. This medium was inoculated with a 3-mm PDA plug of R. solani and incubated in the HPIS for 7 d in the dark at 25°C. The lid of compartment A was sealed with a caulking cord material to prevent the fungus from growing out of the compartment. Modifications for the host compartment Β were accomplished by removing the false bottom of compartment A. A plastic grid was incorporated into the Lutri-plate for support of the medium once the false bottom was removed. A sterile, 86mm membrane (0.2 μτη pore) was sealed in place using nontoxic glue on the bottom side of the water agar-plastic grid. A sterile tissue culture medium containing 3 mg L" 2,4dichlorophenoxyaeetic acid (2,4-D) (2) was then poured into compartment Β of the HPIS. Once the tissue culture medium was solidified, the membrane was sandwiched between the growth media, serving as a physical barrier, restricting the fungus to compartment A . 1

1



316 Complete assembly of the HPIS was carried out in a laminar flow hood to maintain sterility. Callus and plantlet recovery, Penncross callus was co-cultured (period in which callus and fungus were simultaneously growing in the HPIS) with R. solani to determine if surviving callus could be recovered and maintain plantlet regeneration capabilities. Penncross stock callus was obtained according to callus induction and maintenance procedures described by Krans et al. ( i i ) . An 86-mm glass fiber support disc was placed on the top surface of the tissue culture medium contained in compartment Β of the HPIS. Stock callus was plated onto discs at the rate of 0.3 g, with callus aggregates ranging between 0.25-0.5 mm (2). Compartment A of the HPIS, the pathogen side, contained either a highly virulent isolate (RVPI) or a weakly virulent isolate (R12) of R. solani as a treatment. Based on preliminary HPIS experiments, (data not shown), Penncross callus was co-cultured with RVPI or R12 for 12, 24, 36, or 48 h. Controls were callus populations plated into uninoculated HPIS. Following each co-culture period, callus populations were transferred from HPIS via glass fiber support discs to either determine callus viability or plantlet regeneration. A 2 χ 2 factorial completely random experimental design was used, including twelve replicated plates per treatment; six replicates per treatment in callus viability determinations and six in plantlet regeneration. Callus viability was determined following co-culture periods to quantify survival. This was accomplished by treating callus populations with 3 ml of a 2% 2,3,5 triphenyl tetrazolium chloride (TZ) vital stain. To measure callus viability, a transparent, circular grid (47-mm diam.) comprised of 50 random sites (2 mm ) was designed to fit on the cover of a Petri dish (90-mm diam.). The grid-cover was placed over a Petri dish containing a co-cultured callus population. A n initial count of callus tissue in random sites was determined and then treated with 3 ml TZ. Following a 12 h incubation in the dark, a second survey of the callus populations was performed to determine the number of stained callus in the random sites. Callus viability was calculated using the formula: 2

% viable callus = no. stained callus sites χ 100 initial callus count Plantlet regeneration was evaluated on callus that had co-cultured 12,24,36, or 48 h with R. solani isolates in the HPIS. Following each co-culture period, callus populations were allowed to regenerate in plantlet regeneration medium that was similar to the tissue culture medium used in the HPIS but without 2,4-D. To induce plantlet regeneration, callus was maintained under continuous cool white flourescent light (41 μπιοί m V ) at 25°C. The number of plantlets was recorded at the two-leaf stage. Callus viability and plantlet regeneration were analyzed using analysis of variance procedure of Statistical Analysis Systems (SAS), mean separation was carried out using Fisher's least significant difference (P< 0.05) test (25). 1

Screening of R. solani selected and non-selected Penncross plantlets. The HPIS was used to recover isolate RVPI-selected and non-selected plantlets. To obtain RVPIselected plantlets, Penncross stock callus was plated in the HPIS as previously described



317 and co-cultured 24 h with isolate RVPI. Following co-culture, callus populations were transferred to regeneration medium and cultured for plantlet regeneration as previously described. Non-selected plants were derived from callus populations co-cultured in the HPIS without fungus. Selected and non-selected treatments were replicated 5 times. A l l plantlets were transferred and maintained (subcultured every 5 wks) in Magenta boxes containing 25 ml regeneration medium under the same culture conditions as mentioned above. Selected and non-selected plantlets served as stock material in the evaluation of the three in vitro bioassays. The HPIS was assembled as previously described with isolate RVPI growing in the pathogen compartment A . Six segments 20 to 30-mm long consisting of a single node, were harvested from selected and non-selected plantlets. Node sections were placed into the host compartment Β of the HPIS and co-cultured 15 d under the same environmental conditions described for plantlet regeneration. Disease was rated at the end of the coculture period using a visual scale of 0 to 5; where 0=no necrosis, and 5 = total necrosis. Controls developed to establish a baseline for necrosis included nodes that were incubated in the HPIS with no fungus present. A completely random design included nodal tissue of RVPI-selected and non-selected plantlets replicated 30 times. Disease ratings were analyzed using analysis of variance procedure of SAS and comparisons were determined by an F-test (23). Extract Bioassay. An extract bioassay consisted of screening RVPI-selected and nonselected stock plantlets, based on plant response to extracts obtained from liquid cultures inoculated with R. solani isolate RVPI. Culture preparation. Treatments consisted of liquid media with leaf tissue, shredded wheat, or potato added as the carbon source. Isolate RVPI was the inoculum source, and liquid cultures were incubated 21 d at 25°C under continuous cool white fluorescent lights. The control contained sterile, distilled water. Liquid cultures consisted of Murashige and Skoog's basal medium (MS) (17) including 2.2 g salts, 30 g oven-dried, finely ground bentgrass leaves, 30 g finely ground shredded wheat, or 24 g potato dextrose broth (PDB) incorporated into one liter of distilled water. The pH was adjusted to 5.8, and the medium was autoclaved at 121°C at 100 psi for 15 min. After cooling, 1 ml of MS vitamins was added to media except that containing PDB. The media were inoculated with one mycelial plug each from a culture of isolate RVPI growing on PDA. Extraction procedure. The extraction procedure, as reported by Mikel (16), was used to obtain the initial extracts. Liquid cultures were extracted by thoroughly blending the contents then neutralizing with sodium acetate or sodium hydroxide. Liquid cultures were extracted by initial mixing with 0.1% ethyl acetate (ratio of the liquid culture volumes to ethyl acetate, 1:3) in separatory funnels with agitation to facilitate extraction. A liquid and solid phase resulted, and the liquid phase was subjected to three additional extractions using equal volumes ( 1:1 ) of ethyl acetate. The final extract was dry-filtered over anhydrous sodium sulfate and absorbent cotton in a Buchner funnel placed in a


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vacuum-adapted flask. The precipitate was further dried in a rotation evaporator at 55°C. Precipitates were dissolved in 10 μΐ of acetone and distilled water containing a 0.1% surfactant to give a final volume of 1 mg extract ml ~*. Solutions were then sonicated 1520 min in a Fisher Scientific FS 15 Sonicator to promote dissolving of extracts. Bioassay. Extract evaluations were conducted on RVPI-selected or non-selected plantlets. A 24 well (20 χ 30 mm) tissue culture plate contained a filter paper disc (20mm-dia) that supported a single node harvested from each plantlet source. Two hundred microliter aliquots of each treatment extract was dispensed into wells containing nodal tissue. Saturated filter paper facilitated prolonged exposure of nodes to the extracts. The plates were covered with transparent lids to minimize evaporation of extracts. Selected and non-selected nodes were incubated with extract treatments in the light at 25°C for 6 days. Following incubation, disease was evaluated using a scale of 0 to 5 where 0 = no necrosis and 5 = total necrotic tissue. A completely random experimental design included 30 replicates per treatment for each node source. Disease ratings of RVPI-selected and non-selected nodes were analyzed using analysis of variance procedure of SAS and comparisons were determined by an F-test (23). Direct Bioassay. RVPI-selected and non-selected plantlets were exposed to R. solani isolate RVPI by direct inoculation. Stem segments, 20 to 30 mm long, consisting of a single node were harvested from each plantlet source. Nodal tissue was placed onto tissue culture medium previously described in this chapter. Each node was inoculated with a plug of RVPI mycelium by placing the plug directly adjacent to the node. Inoculated tissues were incubated 15 d in continuous cool white flourescent light at 25°C. Controls consisted of a node inoculated with a PDA plug, with no fungus present. Following incubation, disease ratings were recorded based on a scale of 0 to 5 where 0 = no necrosis and 5 = total necrosis. A completely random experimental design was used including 30 replicates for each node source. Disease ratings were analyzed using analysis of variance procedure of SAS and comparisons of injury was determined by an F-test (23). Results and Discussion Callus and plantlet recovery. Recovery of Penncross callus inoculated with isolate RVPI was significantly reduced (P< 0.05) over all co-culture periods (46%) as compared with either R12 (86%) or the uninoculated control (87%). Callus viability was significantly reduced after each period when co-cultured with RVPI compared to R12 or uninoculated control treatments (Table I). Callus viability in RVPI treatments decreased significantly, with a reduction of 75% observed across the 12 to 48 h period of co-culturing (Table I). As co-culture periods increased with RVPI, callus viability decreased in comparison to callus viability in R12 (10%) and uninoculated control (2%) treatments (Table I). RVPI may produce fungal exudates that exert a certain level of selection pressure on the callus. Callus viability in treatments with R12 that were co-cultured for 48 h was significantly reduced compared to the 12 h co-culture (Table I). Callus viability decreased over an extended exposure period to the weakly virulent isolate. An increased co-culture time may be required to


319

Table I. Percentage of viable bentgrass callus recovered from co-cultures with R. solani isolates in the Host-Pathogen Interaction System. Treatment 12hî 24h 36h 48h % viability RVPI &lA*ë 40Ab 42Ab 20Ac R12 95B a 74B c 87B ab 85B b Control 92B a 80Bb 87B ab 90B a *RVPI = highly virulent and R12 = weakly virulent R. solani isolates; control = no fungus. *Co-culture periods. Callus viability within co-culture periods followed by the same capital letter do not differ according to Fisher's least significant difference (P< 0.05) test. ^Callus viability within treatments followed by the same lower case letter do not differ according to Fisher's least significant difference (P< 0.05) test.


1

§

accumulate inhibitory levels of fungal exudates in the HPIS for weakly virulent isolates. Callus viability in uninoculated control treatments were similar across co-culture periods except at 24 h due to poor vigor of stock callus (Table I). Exposing callus at a level of undifferentiation to fungal exudates produced by a virulent pathogen has been shown to result in successful recovery of plants with enhanced disease resistance (10,24). The HPIS bioassay permits transfer of fungal exudates to a callus culture during concurrent growth (5). RVPI significantly reduced callus viability, and the number of regenerated plantlets was significantly reduced as compared to the uninoculated control (Figure 1). Penncoss callus co-cultured with RVPI up to 36 h maintained the ability to regenerate plantlets at a low frequency. Results of similar in vitro selection studies have demonstrated a decreased plantlet regeneration frequency when the selection pressure threshold was high (3). There were no differences in callus vigor and plantlet regeneration among treatments at the 24 h co-culture period. Screening of J?, solani selected and non-selected Penncross plantlets. RVPIselected and non-selected plantlets were screened with RVPI in the HPIS to determine whether desirable variants could be recovered with enhanced fungal tolerance. Following 15 d co-culture with RVPI, nodal sections of RVPI-selected and non-selected plantlets were rated for necrosis. Control nodes had no necrosis and were not included in the statistical analysis for comparison. RVPI-selected nodes resulted in significantly less necrosis compared to non-selected nodes (Table Π). Based on these results, it was concluded that plantlets derived from RVPI-selected callus were less sensitive to fungal exudates than non-selected plantlets. Additional research must be conducted to definitively demonstrate HPIS can be used as a rapid in vitro bioassay for recovering plants with enhanced resistance to R. solani. It has been demonstrated by other researchers that HPIS can be used as a rapid in vitro bioassay for determining deleterious effects of fungal exudates on seedlings of leguminous species (5,20) and detecting deleterious rhizobacteria for potential biological controls of leafy spurge (Euphorbia esula L.) (27).



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Co-culture period Figure 1. Number of plantlets of creeping bentgrass regenerated from callus populations co-cultured 12 to 48 h with Rhizoctonia solani isolate RVPI, R12, or uninoculated control in the HostPathogen Interaction System. TPlantlet means within a co-culture period followed by the same letter do not differ according to Fisher's least significant difference (P < 0.05) test.


321 Table Π. Tissue necrosis ratings of RVPI-selected and non-selected nodes of creeping bentgrass following a 15 day co-culture with Rhizoctonia solani isolate RVPI in the Host-Pathogen Interaction System Nodal source* Necrosis rating* RVPI-selected

2.8

Non-selected

4.4*


f

RVPI-selected nodes harvested from plantlets regenerated from callus co-cultured 24 h with RVPI in the HPIS. Non-selected nodes harvested from plantlets regenerated from uninoculated callus co-cultured in the HPIS. •Rating based on a visual scale of 0 to 5; where 0 = no necrosis and 5 = total necrosis. *Means differ significantly at Ρ < 0.05.

Extract Bioassay. Extracts of liquid cultures of R. solani were used to screen fungal tolerance in RVPI- selected and non-selected nodal tissues. Nodes were incubated with three RVPI-derived extracts for 6 d. RVPI-selected nodes displayed significantly less necrosis within each extract treatment compared to nodes from non-selected plantlets (Table ΠΙ).

Table ΙΠ. Tissue necrosis ratings of RVPI-selected and non-selected plantlet nodes of creeping bentgrass following a six dav exposure to RVPI-derived extracts Bentgrass leaf tissue Shredded wheat Potato dextrose

RVPI-selected Non-selected RVPI-selected Non-selected RVPInselected Non-selected

1.5 2.9* 0.8 2.2* 0.7 3.4*

+

Extracts derived from liquid cultures of Rhizoctonia solani containing ground bentgrass leaves, ground shredded wheat, or potato dextrose as the carbon source. *RVPI-selected nodes harvested from plantlets regenerated from callus co-cultured 24 h with RVPI in the HPIS. Non-selected nodes harvested from plantlets regenerated from uninoculated callus co-cultured in the HPIS. Rating based on a visual scale of 0 to 5; where 0 = no necrosis and 5 = total necrosis. * Within extracts, non-selected nodes differ significantly at Ρ < 0.05 compared to RVPIselected nodes. 8

Control nodes did not manifest necrosis and were not included in the statistical analysis of comparisons. Based on results of the extract bioassay, fungal extracts may be employed as a selection agent in an in vitro bioassay to successfully discriminate between resistance and susceptibility among plants. Several researchers (4,13,14) have successfully used toxin extracts or culture filtrates in bioassays to recover plants with improved resistance to pathogens.


322 Direct Bioassay. Direct inoculation of nodal tissues harvested from RVPI-selected and non-selected plantlets was conducted to determine whether this method was an effective rapid in vitro bioassay. Nodal tissue from RVPI-selected plantlets displayed significantly less necrosis compared to nodes of non-selected plantlets (Table IV).


Table IV. Tissue necrosis ratings of RVPI-selected and non-selected nodes following a 15 dav direct inoculation/incubation with RVPI. Nodal source* Necrosis rating* RVPI-selected

2.7

Non-selected

4.3*

f

RVPI-selected nodes harvested from plantlets regenerated from callus co-cultured 24 h with RVPI in the HPIS. Non-selected nodes harvested from plantlets regenerated from uninoculated callus co-cultured in the HPIS. *Rating based on a visual scale of 0 to 5; where 0 = no injury and 5 = total necrosis. *Means differ significantly at Ρ < 0.05. Control nodes displayed no necrosis due to harvesting. The direct inoculation approach was effective in the differentiation of RVPI-selected and non-selected nodes. However, this method was considered the least desirable among the three in vitro bioassays. Fungal mycelium, growing saprophytically throughout the culture plates, obscured the nodes and made visual assessment difficult. Summary Environmental awareness over the past decade has increased concern over fungicide use in golf course environments. In 1991, the United States Golf Association initiated funding for research projects aimed at several environmental aspects in a golf course setting, including alternative pest management (8). A need has been identified within alternative pest management for rapid in vitro bioassays to select and screen turf grasses with enhanced resistance to pathogenic fungi. Three in vitro bioassays discussed in this chapter were used to discriminated between pathogen-selected and non-selected plantlets. RVPI-selected plantlets were derived from callus exposed to isolate RVPI at the level of undifferentiation. A reduction in plant necrosis was observed in all in vitro bioassays when RVPI-selected nodes were screened with the pathogen. Based on these results, in vitro bioassays can be used to select and screen for enhanced resistance to R. solani in creeping bentgrass.

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Fate and Management of Turfgrass Chemicals - American Chemical

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