Canadian Journal of Plant Pathology Etiology of basal ...

This article was downloaded by: [Montana State University Bozeman] On: 24 March 2015, At: 10:13 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Canadian Journal of Plant Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tcjp20

Etiology of basal kernel blight of barley caused by Pseudomonas syringae pv. syringae a

b

b

C. Martinez-Miller , A. Braun , S.L. Siemsen & D.C. Sands

b

a

EcoPharm, a Division of Pharmagenesis , 900 Technology Blvd, Bozema, MT, 59718 b

Department of Plant Pathology, Montana State University , Bozeman, MT, 59717 Published online: 23 Dec 2009.

To cite this article: C. Martinez-Miller , A. Braun , S.L. Siemsen & D.C. Sands (1997) Etiology of basal kernel blight of barley caused by Pseudomonas syringae pv. syringae , Canadian Journal of Plant Pathology, 19:4, 337-346, DOI: 10.1080/07060669709501057 To link to this article: http://dx.doi.org/10.1080/07060669709501057

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Canadian Journal of Plant Pathology Revue canadienne de phytopathologie Published by

Publiée par

The Canadian Phytopathological Society

La Société Canadienne de Phytopathologie

Volume 19(4):337^J29, i-xiv

December

1997

decembre

ISSN 0706-0661

CANADIAN JOURNAL OF PLANT PATHOLOGY 19:337-346, 1997

Downloaded by [Montana State University Bozeman] at 10:13 24 March 2015

Etiology of basal kernel blight of barley caused by Pseudomonas syringae pv. syringae C. Martinez-Miller, A. Braun, S.L. Siemsen, and D.C. Sands EcoPharm, a Division of Pharmagenesis, 900 Technology Blvd., Bozeman, MT 59718; and (A.B., S.L.S., D.C.S.) Department of Plant Pathology, Montana State University, Bozeman, MT59717. Corresponding author, D.C. Sands. Accepted for publication 1997 09 25

Basal kernel blight is a barley disease that primarily impacts the malting and brewing industry since microorganisms present in diseased kernels may alter malt and beer quality. Basal kernel blight is characterized by dark brown discoloration on the embryo end of the kernel, and in Montana this disease is caused by Pseudomonas syringae pv. syringae. In this study we determined that the critical period of infection is from late milk to soft dough stages of kernel development and that high moisture is necessary during this period of infection and disease development. In addition, the use of sprinkler irrigation at soft dough may be applicable for evaluating susceptibility of cultivars under field conditions as statistical differences were found between cultivars. Martinez-Miller, C , A. Braun, S. L. Siemsen, and D.C. Sands. 1997. Etiology of basal kernel blight of barley caused by Pseudomonas syringae pv. syringae. Can. J. Plant Pathol. 19:337-346.

La bruiure bactérienne est une maladie de 1'orge dont les inconvénients sont principalement ressentis dans les industries du maltage et du brassage puisque les microorganismes dans les grains malades peuvent altérer la qualité du malt et de la bière. La bruiure bactérienne est reconnaissable a la coloration brun foncé que prend l'extrémité du grain oü se trouve l'embryon. Au Montana, cette maladie est causée par Ie Pseudomonas syringae pv. syringae. Dans la présente étude, nous avons determine que la période critique pour l'infection s'étendait de la fin du stade laiteux du développement du grain jusqu'au stade pateux mou et qu'un taux élevé d'humidité était nécessaire durant cette période d'infection et de développement de la maladie. De plus, l'arrosage par aspersion au stade pateux mou peut être utilise pour évaluer la sensibilité des cultivars dans des conditions de champ puisque des differences statistiques entre les cultivars ont été trouvées. Kernel blight, bacterial kernel spot, black point, and kernel discoloration describe symptoms on barley kernels from diseases caused by bacteria or fungi which are common in barley growing areas worldwide ( 1 , 3 , 19, 23). Barley kernel blight has been reported to be predominantly associated with fungi in the Upper Midwest of the US (6,21). Cochliobolus sativus, a common causal agent of kernel blight, was found to be the most prevalent fungal pathogen for many years in the Midwest but has been replaced most recently by epidemic outbreaks of fusarium head blight (20). Alternaria alternata, Cladosporium herbarum, and Arthrinium arundinis can also cause kernel blight but are of minor importance (15,19). Barley kernel blight has also been associated with bacteria in Idaho and Montana (16, 17,18,24). Pseudomonas syringae [van Hall 1902] pv. syringae [v. H a l l ] was the first b a c t e r i u m described causing bacterial kernel spot of Klages barley grown under irrigation in Idaho (24). This symptom has also been reported in Montana and

develops as well defined discoloration with distinct margins on the lemma of the kernel (17). Another, more commonly found disease symptom in Montana is basal kernel blight, which consists of dark brown discoloration at the embryo end of the kernel (17). Similar symptoms have been associated with bacteria by Basson et al. (4), who found counts of a Bacillus spp. on black-end barley kernels significantly higher than on healthy kernels. Furthermore, Toben et al. (27) reported basal glume rot on barley caused by Pseudomonas syringae [v. Hall] pv. atrofaciens f(Mc C u l l o c h ) Y o u n g et al.] in W e s t Germany, and in Russia this bacterium has been reported to cause basal bacteriosis on barley, rye, and wheat (8,12). The malting and brewing industries prefer barley free of kernel blight and will discount, or even reject, discolored barley when blight percentages exceed 4%. Kernel blight reduces grain yield and quality (1), and Gebhardt et al. (6) reported that protein, malt extract, wort color, beer taste and

337


338

CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997

aroma were affected by kernel discoloration. Basson et al. (4) indicated that bacteria, rather than fungi, could play a role in the suppression of germination and in the formation of the black-ends. In this paper we demonstrate that the bacterium Pseudomonas syringae pv. syringae is the primary causal agent of basal kernel blight symptoms of barley in Montana. We describe the effects of kernel developmental stage, humidity, and temperature on the development of disease symptoms. Furthermore, a method of cultivar screening for genetic resistance was developed and its reliability is discussed. The results from these studies should lead to a better understanding of the impact of P.s. pv. syringaeinduced kernel blight of barley and might help farmers avoid irrigation during critical plant stages, which favors basal blight symptom development. Materials and methods Samples. Ninety-one 500-g samples of barley, containing 3 to 10% of kernels with blight symptoms, were collected from fields grown in Montana, North Dakota, Minnesota, and South Dakota in 1991, 1992, and 1993. Samples were collected in the different localities by Anheuser-Busch elevator operators. A total of 24 samples from Montana were analyzed in 1991, 13 in 1992, and 21 in 1993. Two samples from North Dakota were investigated in 1991, 5 in 1992, and 10 in 1993; one sample from South Dakota was tested in 1991 and 1992, and none in 1993. Fifty kernels with basal blight symptoms (Fig. 1) per sample in 1991 and 10 kernels per sample in 1992 and 1993 were tested for the presence of bacteria and fungi, Fungal isolations. Kernels were surface sterilized in 0.5% (v/v) sodium hypochlorite and 10 % (v/v) ethyl alcohol for 5 minutes followed by three rinses in sterile distilled water. Kernels were placed onto potato dextrose agar (PDA) amended with 100 ppm streptomycin sulfate. The identity and frequency of fungi isolated was determined after 7 days incubation at 22 ± 2°C. Bacterial isolations. Preliminary isolations using Wilbrink's medium (2) for Xanthomonas campestris pv. translucens, indicated that this bacterium was not present in kernels with basal blight. Thus the majority of this work was done using KBC (King's B medium plus boric acid and cephalexin) a semi-selective medium for P. s. syringae (22). Kernels were individually soaked in 1 mL of sterile phosphate buffer solution (PBS) pH 6.5 (0.5M potassium phosphate, 0.75% sodium chloride) for 2 h at 4°C, then transferred to a rotary shaker at 250 rpm at 23 ± 2°C for an additional 30 min. The suspension was diluted tenfold in PBS and 0.1 mL portions were spread onto KBC plates amended with 75 ug/mL of cycloheximide. Plates were incubated in the dark at 28°C for

48 h. Fluorescent pseudomonads were selected visually for further analysis based on pigment production and/or fluorescence under 366 nm UV light (Model UVL-21, BLAK-RAY, Ultra-Violet Products, Inc., San Gabriel, California). Putative P. syringae isolates were identified according to standard tests, including oxidase reaction (13), hypersensitivity (HR) on tobacco leaves (Nicotiana tabacum cv. Xanthi) (11), and arginine dihydrolase activity (9). The syringae pathovar identification was based on utilization of trigonelline, L-lactate, and quinate by the isolates in addition to the results of tests with BIOLOG (Biolog, Inc. Hayward, CA.) and the Microbial Identification System (MIDI, Microbial ID Inc. Newark, Delaware.). Strains were also tested for the production of syringomycin or its derivatives by the method of Gross et al. (7). Other bacteria and nonfluorescent pseudomonads were also purified and tested for HR on tobacco plants to detect potential pathogens. Fulfillment of Koch's postulates. Thirteen P.s. pv. syringae strains isolated from kernels of different barley cultivars showing typical basal blight symptoms were reinoculated onto cultivar B 2601 in the greenhouse to test their involvement in developing disease symptoms. In addition, three P.s. pv. syringae isolates from hosts other than barley (wheat, cherry, pear), and six other P. syringae pathovars including the cereal pathovars atrofaciens, coronafaciens and striafaciens and the bean pathovars glycinea and phaseolicola were inoculated onto barley heads at soft dough stage to evaluate the ability of these isolates to induce basal blight. Strain descriptions and origins are shown in Table 1. Three barley spikes per pot were tagged and inoculated at the soft dough stage (Zadoks' Growth Stage, ZGS 85) (33) of development, the stage determined as conducive to basal blight development as described below. One pot (19 cm) with three spikes at soft dough stage represented one replication per treatment, and treatments were replicated. All P. syringae isolates were precultured on King's B agar medium (10) for 24 h prior to inoculation. Preliminary experiments using different inoculum concentrations of strain Pss 552 (P.s. syringae isolated from kernels with basal kernel blight of B 2601, MSU collection) at 103, 104, 105, and 107 cfu/mL, resulted in significantly higher percentages of disease with increasing population densities (data not shown). Based on these experiments, a concentration of 107 cfu/mL was used for all following experiments. A bacterial suspension was prepared in sterile distilled water containing 0.025% Tween 20 (polyoxyethylenesorbitan monolaurate). Control plants for this and the following experiments were sprayed with an aqueous solution of 0.025% Tween 20. Inoculum was applied by spraying the spikes using a hand air


MARTINEZ-MILLER ET AL.: BARLEY/BASAL KERNEL BLIGHT

brush (Model Paaschi D500 1/10 H.P.) until runoff (approx. 5 mL per spike). After inoculation, plants were left in a mist chamber providing continuous wetness at 22/18 ± 1°C (day/night) for 48 h before being placed on the greenhouse bench. At harvest, all kernels per replicate were combined. Percentage of kernels with basal blight were calculated based on the weight of diseased kernels divided by the weight of total kernels per replicate. This method of evaluation was chosen since it is commonly used by brewing companies at quality grading stations. A second experiment using a spontaneous rifampicin-resistant mutant of P. s. syringae Pss 552 was conducted to confirm the reisolation of the inoculated strain. Pss 552 rif was inoculated onto six B 2601 spikes at soft dough stage and incubated as described above, while six B 2601 spikes were not inoculated and served as control plants. After harvest, isolations were made on King's B plus 100 ppm rifampicin from five noninoculated symptomless control kernels, five inoculated symptomatic kernels, and five inoculated symptomless kernels. Kernels were individually soaked as described above for bacterial isolations and dilutions were spiral plated (Model C spiral plater, Spiral Biotech, Bethesda, MD) and bacterial numbers calculated as log cfu/kernel, Effect of kernel developmental stage on the onset of basal kernel blight under greenhouse conditions. The barley cultivars B 2601 (six-row) and B 1202 (two-row), provided by Busch Ag Resources, were used to determine the effect of kernel developmental stage on the severity of basal kernel blight. These cultivars are two of the most commonly grown malting barleys in the Fairfield, Montana, area and are considered moderately susceptible to bacterial kernel blight. A two-factorial completely randomized experimental design with three replications per treatment was used, with the factors inoculum and kernel stage at inoculation. Seeds were planted at weekly intervals to permit different kernel developmental stages at time of inoculation. Three spikes per pot were tagged and inoculated with P.s. syringae Pss 552 or water at the four different developmental stages (early milk (ZGS 73) (31), late milk (ZGS 77), soft dough (ZGS 85), and hard dough (ZGS 87)). Plants were then incubated in a mist chamber for 48 h before being returned to the greenhouse bench. The greenhouse and mist chamber were maintained at 22 ± 2°C during the 14 h day period and at 12°C during the 10 h night period. At maturity, tagged heads from each replication were harvested, combined, and assessed for the percentage of basal kernel blight. The experiment was conducted twice. Effect of duration of free moisture and temperature on basal kernel blight under greenhouse conditions. Cultivar B 2601 seeds were planted at

339

weekly intervals in the greenhouse to allow two kernel developmental stages to occur simultaneously at time of inoculation. Plants were tagged and inoculated as previously described with P. s. syringae Pss 552 at early milk (ZGS 73) (31) and soft dough (ZGS 85) stages of development. The experiment consisted of a three-factorial completely randomized design with three replications (a 19 cm pot with three barley heads tagged at the desired stage prior to inoculation) per treatment. The three levels of moisture tested were; no moisture, 24 h in a mist chamber after inoculation, and 48 h in a mist chamber after inoculation. The three temperatures tested were based on average climatological data from Fairfield, MT., where basal kernel blight occurs regularly (17). After inoculation of plants and incubation in the mist chamber, plants were kept at 18°, 22°, or 28 ± 2°C during the day and 14°, 12°, and 22 ± 2°C at night, respectively. All greenhouse experiments were conducted with a 14 h photoperiod. Percentage of kernels with basal blight was calculated on a weight basis as described above. This experiment was conducted twice and data analyzed separately and in combination. Effect of kernel developmental stage and moisture under field conditions. Field experiments were performed in Fairfield, MT, during the growing seasons of 1992 and 1993. Effect of kernel developmental stage on the severity of basal kernel blight was studied by enhancing the disease development with overhead sprinkler irrigation at early milk, late milk, soft dough, and hard dough stages (ZGS 73, 77, 85, 87) in 1992, and early milk, late milk, and soft dough stages in 1993. Effect of moisture was evaluated by comparing disease severity in irrigated versus nonirrigated plots over all stages. The experiments were planted as split-plot designs with six replicates per treatment, each replicate consisting of 6 rows 1.5 m long with 30 cm between rows. Two identical plots were planted, one to be under overhead irrigation and the other without overhead irrigation. Data were analyzed as appropriate for a two-factorial experiment with kernel developmental stage and moisture (irrigation) as the two factors. Seeds of barley cultivars B 2601 and B 1202 were planted four times at weekly intervals in 1992 beginning April 23 and in 1993, due to wetter weather, at only three intervals beginning May 11 to allow plants to be at different developmental stages at the time of irrigation. Fifteen individual spikes per replication were tagged randomly at each kernel developmental stage and irrigation was applied for approximately 4 hours in the morning and 4 hours in the evening for five consecutive days beginning July 28 in 1992 and August 7 in 1993. During irrigation, sprinklers were on for 5 minutes and off for 30 minutes to maintain free moisture on the barley spikes and avoid lodging. A fine uniform mist was generated


340

CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997

by using a solid high pressure irrigation system of impact head sprinklers (Rainbird Sprinklers) delivering an average of 15 mm of water per 4 hour of irrigation period. The nonirrigated plot remained completely dry during the overhead irrigation period (5 days) in 1992. However in 1993, 2 to 3 mm fell on August 8 and 11 providing some moisture to the nonirrigated plot. At maturity, marked spikes were harvested from both plots and percentage of basal kernel blight was determined as described above. Percentages were calculated over both cultivars. In 1994, a two-factorial split-plot experiment was conducted comparing two levels of irrigation, five versus ten days, over three cultivars. Irrigation was started on July 14, when barley heads were at soft dough stage. The tested cultivars were Robust (six-row), B 1215 (two-row), and B 2912 (six-row). The experiment was planted on April 15 in four blocks. Traces of precipitation (0.6 mm) fell from July 14 to July 23. Cultivar evaluation under field conditions at Fairfield, MT. In 1993, six cultivars (three 2-row and three 6-row) were selected based on their previously observed moderate to high susceptibility to bacterial kernel blight. These included the Busch Ag Resources cultivars B 2912, B 3380, B 1202, and B 5133, the commercial cultivar Klages, and the genotype Mn567 from Minnesota, courtesy of R.D. Wilcoxson, which is resistant to kernel discoloration caused by Cochliobolus sativus. Cultivars were planted on May 1 1 and May 25, in a split-plot design with planting dates (kernel developmental stages at time of irrigation) as main plots and cultivars as subplots with three replications (blocks). Each subplot consisted of 3 rows, each 1.5 m in length, with 30 cm between rows. On August 6, 15 spikes per experimental unit were tagged at either early milk or soft dough stages. Beginning on August 7, overhead irrigation was provided for five consecutive days as described previously. At maturity, marked spikes were harvested, and percentage of basal blight determined. In 1994, 12 cultivars were planted as subplots in a two-factorial split plot design, on May 3 and 12, with developmental stage at irrigation being the main plots. The experiment had four replications (blocks). Irrigation was provided for five days beginning July 19 and disease evaluation was performed in the same manner as in previous years. Traces of rain (0.6 mm) fell during the irrigation period on July 19. The experiment was repeated in 1995, when cultivars were planted on May 3 and 17. In contrast to 1994, irrigation was commenced on July 21 and extended for 10 days to increase the disease incidence and facilitate the screening process, taking into account the data from 1994. A total of 16 mm rain fell during the irrigation period. The experiment was harvested

and evaluated as in previous years. Regular irrigation, soil management, fertilizer application, and herbicide treatments before and during the growing season were done in all years according to common agricultural practices by farmers in the area. All statistical data analyses were performed as appropriate to the experimental design using the MSUSTAT program (14). Data were analyzed by analysis of variance (ANOVA), and the sources and amount of variation were compared using an F test. To compare differences between treatment means, least significant differences (P < 0.05) were calculated. Results and discussion Bacterial and fungal isolations. P. syringae was the only pathogenic bacterium isolated from kernels with basal kernel blight symptoms (Fig. 1). All non-P. syringae isolates found in symptomatic kernels were negative for HR on tobacco and were identified as saprophytes; these included Erwinia herbicola, Erwinia spp., and some Clavibacter sp. Two major groups of P. syringae were differentiated: one group producing syringomycin-like toxins, a trait indicative of P. s. syringae (25) and the other group not producing any detectable antifungal metabolite. This latter group of bacteria had characteristic carbon source utilizations indicative of P.s. coronafaciens but, unlike the latter pathovar, isolates in the group do not produce tabtoxin. The presence of this group of bacteria on blighted kernels was of minor importance and is discussed elsewhere (17,18). All toxin-producing isolates were confirmed to be P.s. syringae based on trigonelline, L-lactate, and quinate utilization, in addition to BIOLOG and MIDI identification. Furthermore, these isolates hybridized to an internal segment of the syrB gene, involved in syringomycin synthesis (17,18). These strains were present in 90 to 100% of the kernels with basal blight symptoms in Montana samples during the three years of evaluation (Fig. 2). In the samples obtained from the other states, however, P. s. syringae was isolated from only 5 to 30% of the diseased kernels in 1991 and 1993, and in 20 to 75% of symptomatic kernels in 1992 (Fig. 2). P. s. syringae has never been considered a problem in the Dakotas or Minnesota. However, our data show that in wet years, such as 1992, P.s. syringae did occur at high frequencies in grain samples (Fig. 2). On the other hand, fungi previously reported to cause kernel discoloration of barley, such as Cochliobolus sativus and Alternaria sp., were found in the Montana samples in lower frequencies than P.s. syringae (Fig. 2). Their low frequency in samples in addition to the bacterial presence suggest that fungi are not the primary cause of this disease in Montana. Koch's postulates and causal organism. A total of 13 P. s. syringae isolates from different barley cul-

MARTINEZ-MILLER ET AL.: BARLEY/BASAL KERNEL BLIGHT

341


The reisolation of the spontaneous rifampicinresistant mutant of Pss 552 from inoculated, but not from noninoculated kernels confirmed Koch's postulates. Pss 552 rif was reisolated at about log 8 cfu/kernel from blighted kernels, while inoculated kernels without symptoms had log 6 cfu/kernel (data not shown). This suggests that disease development may depend on population densities of P.s. syringae and that less efficient multiplication of the bacterium probably results in symptomless kernels.

100

Figure 1. Basal kernel blight symptoms on barley cultivar B 1202 (arrow) inoculated with Pseudomonas syringae pv. syringae Pss 552 at soft dough stage under greenhouse conditions.

tivars exhibiting basal blight were inoculated onto cultivar B 2601 at soft dough stage (Table 1). Although the majority of strains demonstrated a positive HR reaction on tobacco, there were exceptions, such as strain Pss 788. We were unable to correlate level of toxin production in our plate bioassay with disease incidence, but all barley isolates were toxin producers. Therefore, toxin production may contribute to virulence rather than to pathogenicity. These findings are in agreement with those of Xu and Gross (30), who found smaller necrotic lesions on immature cherries using a toxin-minus Tn5 mutant as compared to the toxin-plus wild type. Our isolates produced typical basal blight symptoms in 4 to 63% of all B 2601 kernels inoculated, depending on the strain used. None of the saprophytic non- P. syringae isolates inoculated onto barley heads at the critical stages caused kernel blight symptoms. The inoculation of other cereal P. syringae pathovars such as pvs. striafaciens, coronafaciens, and atrofaciens at soft dough stage revealed that pvs. striafaciens and coronafaciens did not produce basal blight symptoms, while pv. atrofaciens isolates from wheat and barley produced between 8.8 to 20.2% infection on the susceptible host B 2601 (Table 1). The symptoms obtained on kernels after P. s. syringae or P. s. atrofaciens inoculation were indistinguishable. These results are not surprising considering the similarity between the two pathovars in carbon source utilization, and that DNA from P. s. atrofaciens isolates hybridized to the internal 1.1 kb syrB fragment of pv. syringae in southern blots and was found to produce syringomycin or its derivatives in NMR and HPLC studies (5,8,28).

1992

80 (O