© 2008 Plant Management Network.
Alternaria alternata and Plectosporium tabacinum on Snap Beans: Pathogenicity, Cultivar Reaction, and Fungicide Efficacy
Helene R. Dillard, Professor, and Ann C. Cobb, Research Support Specialist, Cornell University, New York State Agricultural Experiment Station, Geneva 14456
Dillard, H. R., and Cobb, A. C. 2008. Alternaria alternata and Plectosporium tabacinum on snap beans: Pathogenicity, cultivar reaction, and fungicide efficacy. Online. Plant Health Progress doi:10.1094/PHP-2008-1212-01-RS.
Pod-flecking complex (PFC) has become an increasing problem for snap bean production. Pods from several commercial fields in New York and one in Pennsylvania were observed in 2006 with small dark flecks and streaks. Two pathogens were isolated and identified as Alternaria alternata and Plectosporium tabacinum. Isolates varied in aggressiveness, and generally isolates of P. tabacinum caused more disease than isolates of A. alternata. Eight snap bean cultivars were susceptible to A. alternata and P. tabacinum. In greenhouse fungicide efficacy trials pyraclostrobin, chlorothalonil, azoxystrobin, and copper hydroxide provided greater than 43% control of PFC caused by either fungus.
Over 12,000 ha of fresh market and processing snap beans are grown in New York. Pod-flecking complex (PFC) reduces product quality, and whole fields are rejected by markets and processing plants when incidence and severity are high (3% to 6%). Pod flecks sometimes are referred to by consultants and producers as russet, seam rust, spots, or rusty or spotty beans. Two pathogens identified as causal agents of PFC are Alternaria alternata (1,11,15,16) and Plectosporium tabacinum (4). In this manuscript, diseases cited in the literature as “pod spot” (16), “pod flecking” (1), “russet” (4), and “black pod” (14,15) are referred to as pod-flecking complex (PFC).
Severe outbreaks of snap bean PFC occurred in New York and Maryland in 2000, and the causal agent was identified as P. tabacinum (4). These outbreaks occurred near harvest and typically followed rain events usually associated with thunderstorms. In 2006, PFC on processing snap bean pods resulted in losses of 8% to 20% or more in some fields in New York and Pennsylvania. The problem also was observed on fresh market snap beans. The symptoms were similar to, but not always consistent with, those caused by P. tabacinum and included tan, orange, or black discolorations in the suture and/or small dark superficial specks, flecks, or spots (sometimes sunken) on the pod surfaces. PFC symptoms intensified with pod maturity, and were most prevalent mid- to late August following periods of prolonged rainfall or rainfall of high intensity.
In addition to P. tabacinum, a species of Alternaria was isolated from pods collected in 2006, but unlike previous reports of A. alternata, characteristic leaf symptoms were not observed (6,13,16). Processors were unable to remove the PFC symptoms prior to packaging the finished product. High culls and rejection of the greater part of one production field led to increasing grower concern in New York and Pennsylvania.
Objectives of this study were to determine etiology, identify Alternaria isolates to species, characterize susceptibility of snap bean cultivars commonly grown in New York and Pennsylvania to A. alternata and P. tabacinum, and examine fungicide efficacy for PFC control.
Isolations of Pathogens
Symptomatic snap bean pod tissue was collected from fields in New York and Pennsylvania (Fig. 1). Excised pod pieces were surface disinfested for 3 min in 30 ml of 0.525% sodium hypochlorite solution containing one to two drops of 95% ethanol, and rinsed once for 3 min in 30 ml of sterile distilled water (SDW). Alternaria was isolated by placing four aseptically excised pieces (2 mm²) on Difco potato dextrose agar (PDA) (Becton, Dickinson, & Co., Sparks, MD) amended with streptomycin and chloramphenicol (each 50 mg/liter) in Petri plates (100 × 15 mm) (ABPDA). Plectosporium was isolated from diseased areas on pods, triturating tissue in a drop of SDW on a microscope slide, and streaking on ABPDA (4). All plates were incubated at 21 to 24°C with 14 h of cool white fluorescent light (25 microeinsteins/m²/sec). Ten isolates of A. alternata and four isolates of P. tabacinum obtained in 2006 were used in this study (Table 1).
Table 1. Characteristics of isolates of Alternaria alternata and Plectosporium tabacinum isolated from snap bean pods in 2006.
x New York unless specified otherwise.
Identification of A. alternata Isolates
Genomic DNA was extracted from 1- to 2-week-old fungal cultures of representative bean isolates 1058, 1060, 1062, 1070, 1080A, and 1089 using the UltraClean Soil DNA Isolation Kit (MoBio, Carlsbad, CA). Extracted genomic DNA concentrations ranged from 15 to 120 ng/mL. Internal transcribed spacer (ITS) regions of the fungal isolates were amplified with primers ITS5 and ITS4. Polymerase chain reaction (PCR) was carried out with 1 × ThermoPol reaction buffer (New England BioLabs, Ipswich, MA), 1 mM dNTPs, 0.2 mM primer each, 1 unit Taq DNA polymerase (New England BioLabs, Ipswich, MA), and 15 ng of genomic DNA in a 50 mL reaction. PCR cycling conditions were: 94°C for 5 min, 35 cycles of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C, followed by 10 min at 72°C. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Samples were sent to Cornell University Life Sciences Core Laboratories Center, Ithaca, NY for sequencing. The ITS sequences for the isolates tested were identical to A. alternata and A. tenuissima when BLAST searched in the GenBank (9,10). Furthermore, the most parsimonious tree (bootstrap value = 100) grouped the isolates with A. alternata, A. arborescens, A. longpipes, and A. tenuissima (7,9,10,17). Following a review of the existing literature, we determined that our isolates best fit the description of A. alternata on the basis of morphology and host range (5).
Production of Inoculum
After comparing and modifying various methods for production of A. alternata inoculum (1,2,3,8,11,12), the following method was devised for use in these studies. Three 5-mm diameter agar disks from colonies of each A. alternata isolate were placed equidistant on PDA in Petri plates and incubated at 21 to 24°C with 14 h of cool white fluorescent light for 4 days to produce conidiophores (Fig. 2A). The isolates were transferred as above to 50% PDA and placed in an incubator (24°C) in the dark for 13 to 20 days to induce sporulation. Conidia were harvested by flooding Petri plates with SDW, scraping the culture with a rubber policeman (Fisher Scientific, Pittsburgh, PA), and filtering the resulting spore suspension through sterile cheesecloth.
P. tabacinum isolates were streaked on PDA plates and incubated at 21 to 24 °C with 14 h light for 6 to 9 days (Fig. 2B). Spores were harvested by flooding plates with SDW.
Snap bean seeds were planted (four to five seeds per pot, thinned to three plants) in square pots (121 cm²) filled with Cornell mix (4) and grown in the greenhouse at 20 to 25°C. The experimental unit was one pot, with three replications per treatment. Spore suspensions (5 ml/pot) of each isolate of A. alternata or P. tabacinum were atomized onto leaves and marketable size pods using a Preval (Precision Valve Corporation, Yonkers, NY) sprayer 49 to 61 days after planting. Conidia of A. alternata isolates 1058, 1061B, 1062, 1064, 1070, 1080A, and 1089 were applied at 105 spores/ml. A. alternata isolates 1059, 1060, and 1069 were applied at 106 spores/ml (these isolates consistently produced more spores). All isolates of P. tabacinum were applied at 107 spores/ml in all experiments. After inoculation and air drying 2 h, pots were placed in a mist chamber for 5 days at 23 to 25°C with 14 h light. Continuous fine mist (fog) was generated using pneumatic nozzle assemblies (Spraying System Co., Wheaton, IL) with 2.8 kg/cm² air pressure (Fig. 3).
Eighteen A. alternata isolates were tested on processing snap bean cultivars GoldMine, Hystyle, and Diplomat, and Koch’s postulates were satisfied by re-isolation from symptomatic tissue. A subset of ten A. alternata isolates was selected to represent the greatest possible diversity of location, symptoms, appearance of the cultures on PDA, and spore-producing ability. Four P. tabacinum isolates were also evaluated (Table 1). Isolates of both pathogens were tested for pathogenicity on cultivars GoldMine, Hystyle, Diplomat, Titan, and Bronco, and the experiment was repeated.
The first symptoms appeared 3 days after misting and the plants were rated 2 days later (Fig. 4). Disease severity increased over time, with more flecks on the pods and enlargement of existing flecks. Severity was visually estimated and evaluated as percent diseased tissue of all individual mature pods averaged per pot. Severity values were transformed using the arcsin square root transformation for all experiments. Transformed data were analyzed using PROC GLM (SAS Institute Inc., Cary, NC). Incidence [(number of infected pods / total number of pods per pot) * 100] was evaluated in all experiments. Virtually every pod had at least one fleck by 5 days post inoculation; therefore severity was deemed the most effective measurement of pathogen aggressiveness.
Aggressiveness of isolates. There were significant cultivar*run interactions and data were separately evaluated for effects of pathogen (i.e., two species), isolates within pathogen, and cultivar. Significant cultivar*pathogen and cultivar*isolate within pathogen interactions were found. All isolates were pathogenic and varied in aggressiveness (Table 2). In general, P. tabacinum caused more disease than A. alternata. No disease occurred in non-inoculated controls.
Table 2. Pod-flecking severity of ten isolates of Alternaria alternata and four isolates of Plectosporium tabacinum on five snap bean cultivars.
x Values are the mean of three replications.
y Standard error (SE) of the means.
Cultivar susceptibility. To determine if cultivars varied in susceptibility, eight cultivars (four replications) were subjected to pathogenicity tests using A. alternata isolates 1060 and 1070, and P. tabacinum isolates 1055 and 1080P. The experiment was repeated. There were no significant cultivar*run or isolate*cultivar interactions. All cultivars were susceptible to A. alternata and P. tabacinum (Table 3). Titan consistently developed high levels of disease when inoculated with either pathogen. Non-inoculated controls averaged 1% disease severity in run 1 and 1.8% disease severity in run 2.
Table 3. Pod-flecking severity caused by Alternaria alternata and Plectosporium tabacinum on eight snap bean cultivars.
x Data were averaged across isolates 1060 and 1070 (no significant isolate*cultivar interaction); N = 8. Statistics apply within columns.
y Data were averaged across isolates 1055 and 1080P (no significant isolate*cultivar interaction); N = 8. Statistics apply within columns.
z Mean percent disease severity on snap bean pods values are shown. Treatment means were separated using LSD (P ≤ 0.05).
Fungicide Efficacy Tests
Snap bean plants (cv. Titan) and inoculum of A. alternata and P. tabacinum were prepared as previously described. Near pod maturity (56 days after planting), four replicate pots were arranged in a single row. On day 1, a CO2 backpack single row sprayer calibrated to deliver 635 l/ha of water at 345 kPa using three 8002 flat fan nozzles and traveling at 2.9 km/h, was used to apply the fungicide. The sprayer was configured with one nozzle positioned over the top of the row and 22.9-cm drop nozzles positioned on each side of the row angled down. Chemicals were selected based on their reported use in controlling other diseases caused by Alternaria species (Table 4). Plants were air dried for 2 h and returned to the greenhouse. A. alternata isolates 1060, 1070, and 1089 and P. tabacinum isolates 1055 and 1080P were selected to provide pathogen diversity in the efficacy test. On day 2, the plants were inoculated as previously described, air dried for 2 h, and placed in the mist chamber and incubated. Disease incidence and severity were recorded on day 7 as described above and the trial was repeated.
Table 4. Effect of fungicide treatments on snap bean pod disease severity caused by Alternaria alternata and Plectosporium tabacinum.
w NA = Not Applicable.
x Data are means of repeated experiments (cultivar Titan) analyzed across isolates 1060, 1070, 1089; N = 12. Means separated using LSD (P ≤ 0.05). Statistics apply within columns.
y Data are means of repeated experiments (cultivar Titan) analyzed across isolates 1055, 1080P; N = 8. Means separated using LSD (P ≤ 0.05). Statistics apply within columns.
z Vinclozolin is no longer registered for use on snap beans.
Non-inoculated control treatments had no disease. There were no run*isolate and run*fungicide interactions and the two runs were analyzed as blocks. The isolate*chemical interaction was used as the error term in the analysis. The two pathogens were analyzed separately since P. tabacinum caused more disease than A. alternata. Isolates within a species were combined for analysis since there were no significant differences among isolates.
Disease incidence (percent pods infected) was high in all treatments. Incidence of PFC caused by A. alternata was reduced slightly by pyraclostrobin (20%), copper hydroxide (5%), and chlorothalonil (6%) (data not shown). However, no fungicide treatment significantly reduced incidence of PFC caused by P. tabacinum.
All fungicides except vinclozolin significantly reduced disease severity caused by A. alternata (Table 4). The most effective treatments for A. alternata, cyprodinil + fludioxonil, copper hydroxide, chlorothalonil, azoxystrobin, and pyraclostrobin, had less than 10% disease severity on the pods. Iprodione, boscalid, and thiophanate-methyl treatments were intermediate in efficacy.
Disease severity caused by P. tabacinum was significantly reduced by thiophanate-methyl, copper hydroxide, chlorothalonil, azoxystrobin, and pyraclostrobin (Table 4). Vinclozolin, iprodione, boscalid, and cyprodinil + fludioxonil did not significantly reduce disease severity caused by P. tabacinum.
Discussion and Recommendations
In this study, all isolates of A. alternata and P. tabacinum were pathogenic and varied in aggressiveness. In general, isolates of P. tabacinum caused more disease than isolates of A. alternata. The snap bean cultivars tested are currently commercially grown in New York, and all were susceptible to A. alternata and P. tabacinum. The cultivar Titan consistently developed high levels of disease caused by either pathogen.
New York growers speculated that the recent outbreaks of PFC were related to the loss of registration of vinclozolin, previously used on snap beans to control Sclerotinia sclerotiorum and Botrytis cinerea. It was hypothesized that vinclozolin applications suppressed development of PFC caused by A. alternata and P. tabacinum. However, in this study, vinclozolin provided no control of either pathogen. In agreement with Tu’s study on dry beans (14), iprodione reduced disease severity caused by A. alternata. In contrast to Tu’s data, but in agreement with Abawi et al., (1) chlorothalonil reduced the severity of pod-flecking caused by A. alternata. Gomes and Dhingra (6) observed in fungicide field trials that benzimidazole fungicides increased occurrence of symptomatic and nonsymptomatic seed infections by A. alternata, and Abawi et al. (1) found benzimidazole fungicides ineffective for pod-flecking control. In our study, thiophanate-methyl reduced PFC severity caused by A. alternata and P. tabacinum, but other treatments were more effective.
Controlling late season PFC with fungicide applications may not be feasible due to regulatory restrictions on preharvest intervals and the uncertainty of disease development triggered by one or more rainfall events as the pods approach maturity. Appropriate timing of fungicide applications is additionally complicated by A. alternata’s existence as an epiphyte on symptomless dry bean leaf surfaces and weeds, and the population increases as plants age and senesce (14,15). Snap bean producers traditionally make one or two preventative fungicide applications to control S. sclerotiorum and B. cinerea. Selection of fungicides could be made based on the perceived need to control PFC.
The current recommendation in New York is for snap bean producers to harvest at or near peak maturity and avoid harvest delays that would result in overripe pods.
This project was supported in part by the New York Vegetable Research Council and Association and the Pennsylvania Vegetable Marketing and Research Program. We thank farm, industry, extension, and university personnel for their assistance throughout the project.
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