Alternaria alternataandPlectosporium tabacinumon Snap Beans: Pathogenicity, Cultivar Reaction, and Fungicide Efficacy

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. Introduction 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 midto 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. 12 December 2008 Plant Health Progress 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). Fig. 1. Symptomatic snap bean pods from a commercial field in New York (A, cv. Hystyle) and Pennsylvania (B, cv. Diplomat) from which Alternaria alternata was isolated. 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 1to 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. Isolate Countyx Cultivar Symptoms Sporulation on media A. alternata 1058 Genesee Titan suture spots moderate 1059 Orleans Bronco blossom and pod spots profuse 1060 Orleans Bronco specks profuse 1061B Orleans Bronco specks moderate 1062 Orleans Hystyle flecks moderate 1064 Orleans Hystyle suture spots moderate 1069 Orleans Hystyle spots and mycelium on pods profuse 1070 Potter, PA Diplomat sunken lesion moderate 1080A Chautauqua Titan flecks moderate 1089 Orleans Bronco sunken lesion moderate P. tabacinum 1055 Chautauqua Titan specks profuse 1056 Genesee Titan flecks profuse 1057 Genesee Titan flecks profuse 1080P Chautauqua Titan flecks profuse 12 December 2008 Plant Health Progress 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. Fig. 2. Cultures of (A) Alternaria alternata (5 days) and (B) Plectosporium tabacinum (2 weeks) growing on PDA. 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. Pathogenicity Tests 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). 12 December 2008 Plant Health Progress Eighteen A. alternata isolates were tested on processing snap bean cultivars GoldMine, Hystyle, and Diplomat, and Koch’s postulates were satisfied by reisolation 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. Fig. 4. Symptoms of (A) Alternaria alternata from mild to severe, and (B) Plectosporium tabac