Role of co-infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Authors


Abstract

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co-infection of potato. The objective of this study was to determine if co-infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real-time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water-soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co-inoculation with these strains of Vdahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co-inoculated with both pathogens compared to either pathogen alone in both trials, and Vdahliae levels were greater in basal stems of plants co-inoculated with either Pectobacterium isolate. Overall, these results indicate that although co-infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co-infection can increase PED severity.

Introduction

Potato early dying (PED) is a disease complex reported from many potato producing regions around the world including North America, Europe and the Middle East. Fields affected by PED exhibit chlorosis, necrosis, wilting and premature senescence. Several pathogens can be associated with the PED complex including fungi, bacteria and nematodes (Stevenson et al., 1976; Kotcon et al., 1985; Powelson, 1985; MacGuidwin & Rouse, 1990). Verticillium dahliae, the causal agent of verticillium wilt of potatoes, is one of the primary pathogens reported to be involved in the early dying complex of potato in most locations where potato is grown (Rowe et al., 1987). Primary inoculum of V. dahliae consists of soilborne microsclerotia which germinate in response to plant root exudates and colonize the plant xylem resulting in wilt, chlorosis and premature senescence.

The genus Pectobacterium, formerly classified as Erwinia, comprises several species of pectolytic bacteria that cause diseases of potato. Species of Pectobacterium have been reported to cause early dying symptoms similar to those caused by V. dahliae (Kirkland, 1982; Powelson, 1985; Rowe et al., 1987). These bacteria can be associated with soils, water, insects, or the lenticels and surfaces of seed tubers (Silva de Jaczko et al., 1983; Powelson & Apple, 1984; Cappaert et al., 1988). In the Columbia Basin of Washington, USA, Pectobacterium carotovorum subsp. carotovorum is considered to be the most common Pectobacterium species associated with potato diseases, although P. atrosepticum is most often associated with potato blackleg. Pectobacterium wasabiae has also recently been associated with commercial potato production, suggesting a growing role of this Pectobacterium species in soft rot diseases of potato in North America (De Boer et al., 2012). Dickeya, another genus of pectolytic bacteria also previously classified as Erwinia, has been identified as the causal agent of wilt and early dying of potato in Israel (Lumb et al., 1986) and Europe (Toth et al., 2011).

When multiple pathogens are present, interactions may result in additive or synergistic (greater than additive) increases in PED incidence and severity or decreases in yield (Wallace, 1978). Such interactions can also lead to an earlier onset of disease or reduced inoculum thresholds required for disease development (Martin et al., 1982; Rowe et al., 1987). For example, interactions between the root lesion nematode Pratylenchus penetrans and vegetative compatibility group (VCG) 4A isolates of V. dahliae can result in PED symptoms at inoculum levels which individually would not cause disease (Rowe et al., 1985; Rowe & Powelson, 2002). The extent of interactions observed ranged from additive to synergistic depending on initial levels of each pathogen and the dependent variables measured. In addition, these interactions on potato were found to vary depending on the V. dahliae VCG and nematode species involved. The pathogens involved in this disease complex can vary by geographic region and interactions between other nematode species and V. dahliae have been implicated in PED in Florida, USA (Weingartner et al., 1974) and Israel (Siti, 1979).

In addition to PED, pectolytic bacteria can cause soft rot of aerial potato stems, which is known as bacterial stem rot or aerial stem rot (ASR). Unlike blackleg, ASR originates in the canopy and typically occurs after canopy closure (Johnson et al., 2011). Wounds caused by blowing sand, insects, heavy wind, or mechanical damage are required for infection to occur. Greenhouse studies demonstrated that periods of high relative humidity and subsequent plant wetness, which may increase following canopy closure, can increase the development of ASR (Dung et al., 2010). In addition, logistic regression models of meteorological, soil type and previous crop variables with disease incidence in commercial fields across Washington, USA, indicated that high temperatures, a previous crop of maize and sandy soils can also increase the incidence of ASR (Johnson et al., 2011). Differences in the abilities of Pectobacterium isolates to colonize potato stems and cause ASR have been observed (Rahimian & Mitchell, 1984a) and some Pectobacterium isolates can cause severe ASR symptoms only under conditions of high relative humidity and plant wetness (Dung et al., 2010; Johnson et al., 2011).

Verticillium dahliae and Pectobacterium spp. can individually have significant detrimental effects on potato production, but when these pathogens are present together on potatoes their destructive potential can increase (Zink & Secor, 1982; Rowe et al., 1987). However, it is not entirely clear if additive or synergistic interactions occur when potatoes are infected by both pathogens. Previous studies were often based on correlative data or used inoculation methods that may not necessarily represent what naturally occurs in the field. In a 1982 study conducted in Oregon, USA, it was found that P. carotovorum subsp. carotovorum and Patrosepticum were associated with PED symptoms in commercial fields and the relative contributions of either pathogen were correlated with the previous cropping histories of the fields (Kirkland, 1982; Kirkland & Powelson, 1982). It was observed that Verticillium was associated with early dying in fields after several years of potato production, while Pectobacterium spp. were associated with early dying in fields cropped to potatoes for 1 year or less. Fields with high incidences of both pathogens exhibited increased rates of disease progress compared to fields impacted with either pathogen alone (Kirkland & Powelson, 1981). In addition, root dip co-inoculations with V. dahliae and P. carotovorum subsp. carotovorum or Patrosepticum in the greenhouse resulted in greater PED severity and increased basal colonization of stems by V. dahliae compared to inoculations with either pathogen alone, and the effect was additive (Kirkland, 1982).

Another study reported both additive and synergistic interactions between V. dahliae and P. carotovorum subsp. carotovorum (Rahimian & Mitchell, 1984b). Plants that were co-inoculated with both pathogens exhibited greater chlorosis and stem soft rot compared with plants inoculated with either pathogen alone. However, the interactive effects observed ranged between additive and synergistic depending upon the inoculation method used to introduce Pectobacterium into the plant, the concentration of inoculum and the disease variables measured. Synergistic effects were mostly observed when plants were inoculated by dipping the cut end of a stem into inoculum, which was intended to simulate infection arising from an infected seed piece. The synergistic interaction observed using this method was attributed to the longer duration of co-infection and subsequent interaction between the two pathogens, which allowed for greater symptom development. Significant differences between Russet Burbank and Norgold Russet cultivars were also observed in the study, indicating that potato cultivars may respond differently following co-infection by multiple pathogens.

A preliminary survey to determine the co-occurrence of pectolytic bacteria and V. dahliae in commercial fields in Washington, USA, was carried out during the 2008 growing season. The survey was conducted in fields exhibiting severe PED and ASR symptoms. Pectolytic bacteria were isolated from 86% of V. dahliae-infected Russet Burbank potato stems (authors' unpublished data), suggesting a high potential for interactions between these two pathogens in commercial potato fields. The objective of this study was to test the hypothesis that additive or synergistic interactions exist between V. dahliae and Pectobacterium in the development of PED or ASR of potato. Two isolates of Pectobacterium were used: P. carotovorum subsp. carotovorum isolate Ec101, which causes PED symptoms, and Pwasabiae isolate PwO405, which causes ASR symptoms. A relatively non-invasive procedure was used to inoculate Pectobacterium to aerial stems which attempted to mimic artificial wounding caused by insects or windblown sand. PED and ASR severity were measured in greenhouse assays to determine if additive or synergistic increases in disease severity occurred following co-inoculation with V. dahliae and Pectobacterium spp. compared to inoculations with either pathogen alone. In addition, relative levels of pathogen DNA were measured using real-time quantitative PCR (qPCR) in order to determine if co-inoculation with V. dahliae and Pectobacterium resulted in greater pathogen colonization in potato stems.

Materials and methods

Pectobacterium isolates

Pectobacterium carotovorum subsp. carotovorum isolate Ec101, also known as isolate cc101 (Gross et al., 1991), was collected from potato grown in Oregon, USA, and stored in 20% glycerol at −80°C. Preliminary experiments indicated that isolate Ec101 did not cause ASR symptoms (Dung et al., 2010) but was able to induce wilt symptoms similar to PED (data not shown). Pectobacterium wasabiae isolate PwO405 was collected in 2008 from a potato plant (cv. Russet Burbank) exhibiting ASR symptoms in a field located near Othello, Washington, USA (Dung et al., 2012). Isolates were stored in 20% glycerol at −80°C and grown on nutrient broth yeast (NBY) extract agar for 24 h prior to use. Bacterial cells were grown overnight in 5 mL NBY broth cultures at 28°C with agitation for DNA extractions and inoculum preparation. The identity of isolate Ec101 was confirmed by sequence analysis of 16S rRNA, and aconitase (acnA) and malate dehydrogenase (mdh) genes as previously described (Yap et al., 2004; Pitman et al., 2010; GenBank accession nos. JQ740835 to JQ740837). Isolate PwO405 was previously identified using 16S rRNA, acnA and mdh coding sequences (GenBank accession nos. JQ723958 to JQ723960; Dung et al., 2012). Only two Pectobacterium strains were included in the experiment because the use of additional strains would have greatly increased the number of treatment combinations and samples for processing.

Pathogenicity of Pectobacterium isolates to potato tubers

The ability of isolates Ec101 and PwO405 to cause soft rot of potato tubers was confirmed using a tuber slice assay. Inoculum was prepared from overnight cultures grown as described above. Cells were harvested by centrifugation at 21 380 g for 10 min, washed with sterile distilled water (SDW), and resuspended in SDW to an optical density (OD600) of 0·3 (c. 108 colony-forming units (CFU) mL−1). Certified seed tubers of Russet Burbank potato were surface-sterilized in 0·5% NaOCl for 5 min and allowed to air dry at 10°C for 24 h. Tubers were cut in cross section into slices c. 6–8 cm wide and 1 cm thick using a flame-sterilized knife. Tuber slices were weighed and then inoculated by applying 50 μL inoculum (5 × 106 CFU) to a 1 cm2 piece of sterile Whatman filter paper placed in the centre of the tuber slice. A SDW-inoculated control (50 μL) was also included. Tubers were incubated at 28°C under 90 to 100% relative humidity. Lesions were measured at 24, 48 and 72 h post-inoculation (hpi) and area under lesion progress curves (AULPC) were calculated using the following formula: math formula, where Yi = lesion size at the ith observation, ti = time (days post-inoculation, dpi) at the ith observation and = number of observations. At 72 hpi, rotted tissue was gently removed from the tuber slices, slices weighed, and the ratio of final mass/initial mass of each slice was calculated. Analysis of variance (anova) was performed in sas (v. 9.2; SAS Institute) using proc mixed and multiple pairwise comparisons were performed using Tukey's Honestly Significant Difference test.

Co-inoculation of Pectobacterium and V. dahliae in the greenhouse

The interaction between V. dahliae and Pcarotovorum subsp. carotovorum isolate Ec101 and P. wasabiae isolate PwO405 in the production of PED and ASR of potato was investigated in the greenhouse. The first trial was conducted between 13 October and 1 December 2010 and the second trial was performed between 28 June and 16 August 2011. Single-stem Russet Burbank potato plants were obtained by rooting uncut, surface-sterilized certified seed tubers in Sunshine LC1 peat-based potting mix (SunGro). Sprouted stems, 10–15 cm in length, were gently removed and transplanted into 10 cm square pots containing LC1 potting mix. Each experimental unit consisted of four subsamples, or single-stem plants. Experimental units were replicated four times and arranged in a randomized complete block design in the greenhouse. Diurnal temperatures in the greenhouse ranged from 10°C (evening) to 27°C (day) in the first trial and 7°C (evening) to 26°C (day) in the second trial in order to replicate the large diurnal temperature variations typical during the potato growing season of the Columbia Basin. Plants were grown under natural light and fertilized with 50 mL Miracle-Gro Pro Select 20-20-20 liquid fertilizer (Scotts Miracle-Gro) approximately every 4 weeks.

Plants were inoculated c. 4 weeks after transplanting with either a 50 mL soil drench of V. dahliae isolate 653 (105 CFU cm−3 soil) or a SDW control. Isolate 653 belongs to VCG 4A and was previously shown to be aggressive on potato (Dung & Johnson, 2012). Bacterial inoculations were performed 4 weeks after V. dahliae inoculations in the first trial and prior to the onset of verticillium wilt symptoms. Inoculations were performed 2 weeks after fungal inoculations in the second trial to allow a greater duration of pathogen interaction within the plant. Plants were wound-inoculated using Pectobacterium isolates Ec101, PwO405, or a SDW control. Bacterial inoculum was prepared and quantified as described above and serially diluted to c. 106 CFU mL−1. Stems were wounded by inserting a sterile 23 gauge needle just above a central leaf axil to a depth of 1 mm and a 10 μL drop of inoculum (104 CFU) was immediately placed on the wound. The plants were then placed in a mist chamber and exposed to a 24 h period of leaf wetness at 28–30°C in order to induce ASR symptoms. Plants were removed from the mist chamber and the length of ASR lesions was measured at 24 hpi. Plants were subsequently placed in the greenhouse. Wilt incidence and total leaf chlorosis and necrosis were measured at c. 7, 14 and 21 dpi during the first trial and 14, 21 and 35 dpi during the second trial. Chlorosis and necrosis were recorded as the percentage of plant affected and values were converted to areas under chlorosis progress curves (AUCPC) and areas under necrosis progress curves (AUNPC), respectively, using the following formula: math formula, where Yi = % chlorosis or necrosis at the ith observation, ti = time (dpi) at the ith observation and = number of observations. Chlorosis and necrosis data were combined into areas under senescence progress curves (AUSPC).

Data for each experimental unit consisted of the mean of the four subsamples for each dependent variable (wilt, lesion size, AUCPC, AUNPC and AUSPC). anova was performed in sas using proc mixed. Data were analysed as a two-way factorial design, with treatments consisting of three levels of Pectobacterium (isolate Ec101, isolate PwO405 and a water control), two levels of V. dahliae (isolate 653 and a water control) and trials and blocks as random effects. Data for lesion size were log-transformed to satisfy the assumptions of normality required for anova. A significant statistical interaction between Pectobacterium and V. dahliae treatments in anova would indicate a potential additive, synergistic or antagonistic effect between treatments. Multiple pairwise comparisons were performed using Tukey's Honestly Significant Difference test as described above.

Quantification of V. dahliae and Pectobacterium in potato stems using quantitative PCR

Real-time quantitative PCR (qPCR) was used to detect and quantify V. dahliae and Pectobacterium DNA in Russet Burbank potato stems. Plants from the above greenhouse experiments were sampled by block at 23 dpi in the first trial and 35 dpi in the second trial. Plants were cut at the soil line, surface-sterilized in 0·5% NaOCl for 1 min and allowed to air-dry. Stem cross-sections, c. 1 cm in length, were taken at the inoculation point, 1 cm below the apical meristem and 1 cm above the soil line and processed separately so that pathogen levels could be determined in all three areas.

The four subsamples (single-stem plants) of each experimental unit were bulked for DNA extraction. DNA was extracted from c. 3·5 g of plant tissue using a protocol modified from Khanuja et al. (1999). DNA was purified using the Agencourt AMPure purification system (Beckman Coulter) according to the manufacturer's directions, quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and diluted to 1 ng μL−1 with SDW.

qPCR amplifications were conducted as previously described (Atallah & Stevenson, 2006; Atallah et al., 2007). Quantification of V. dahliae, P. carotovorum subsp. carotovorum, P. wasabiae and potato DNA was performed in separate reaction plates. Each plate consisted of samples from an entire block and all reactions were performed in triplicate using a Bio-Rad iCycler iQ real-time PCR system using previously published annealing temperatures (Atallah & Stevenson, 2006; Atallah et al., 2007). A standard curve consisting of a six point, tenfold serial dilution of V. dahliae DNA was used to enable quantification of between 0·15 pg and 15 ng of V. dahliae DNA, equivalent to between c. 5 and 5 × 105 nuclei for most isolates of V. dahliae (Atallah et al., 2007). A five point, tenfold serial dilution enabled quantification of 10 pg to 100 ng of Pectobacterium DNA. These values are roughly equivalent to between 1·9 × 103 and 1·9 × 107 cells of Pectobacterium, assuming the mpd gene exists as a single copy within the c. 5 Mb genome of Pectobacterium spp. (Glasner et al., 2008). DNA of the potato host was also quantified using a five point, tenfold serial dilution ranging from 5 pg to 50 ng of potato DNA. Standard curves used for quantification of P. carotovorum subsp. carotovorum isolate Ec101 and P. wasabiae isolate PwO405 were generated using DNA from each respective isolate. Standard curves used for the quantification of potato DNA were generated using DNA from a non-inoculated, uninfected Russet Burbank potato plant. A no-template water control was included on all plates. Melt curve analysis was performed to distinguish potential non-specific amplification products.

Cycle threshold (Ct) values, which are inversely proportional to the log concentrations of the target sequences, were averaged among the three replicate qPCR reactions for subsequent analysis. An infection coefficient (IC) for each sample was calculated using the following formula: Ct host/Ct pathogen (Atallah & Stevenson, 2006; Atallah et al., 2007). Larger IC values indicate a greater ratio of pathogen to host DNA. Data were analysed by sample site to identify differences in IC values at basal, apical and inoculation sites among treatments. Data were also averaged over the three sample sites (inoculation point, apical meristem and soil line) to calculate the mean IC values of each experimental unit. anova was performed as a two-way factorial design and tests for significant interactions were performed as described above. Mean V. dahliae and Pectobacterium DNA levels and standard deviations were calculated but not subjected to anova because of violations of assumptions.

Results

Pathogenicity of Pectobacterium isolates to potato tubers

The ability of Pectobacterium isolates Ec101 and PwO405 to cause soft rot on potato slices was evaluated in order to confirm that the lack of ASR symptoms by isolate Ec101 was not due to a loss of overall pathogenicity. Isolates Ec101 and PwO405 caused typical soft rot symptoms of tubers and were capable of macerating tuber tissue within 24 h under the incubation conditions used. After 72 h many tuber slices were completely macerated and only the epidermis, periderm and portions of the cortex remained. The effect of Pectobacterium isolate was significant at < 0·0001 for both AULPC and final mass/initial mass compared to water-inoculated controls in both trials and AULPC values were similar (> 0·05) between isolate Ec101 and isolate PwO405 in both trials (Table 1). Although Pcarotovorum subsp. carotovorum isolate Ec101 caused larger AULPC values in both trials, Pwasabiae isolate PwO405 caused greater tissue loss and this difference was significant in the second trial (= 0·0145).

Table 1. Area under lesion progress curve (AULPC) values and ratios of final to initial mass of potato (cv. Russet Burbank) slices inoculated with two isolates of Pectobacteriuma
Pectobacterium AULPCFinal mass/initial mass
Trial 1Trial 2Trial 1Trial 2
  1. a

    Tuber slices were inoculated with 5 × 106 colony-forming units and incubated at 28°C under 90–100% relative humidity. Values followed by different letters indicate significant differences among treatments within columns using Tukey's test (= 0·05).

Non-inoculated control0a0a0·991a0·992a
P. carotovorum subsp. carotovorum isolate Ec101282b327b0·839b0·779b
P. wasabiae isolate PwO405267b310b0·804b0·689c

Co-inoculation of Pectobacterium and V. dahliae in the greenhouse

Plants inoculated with V. dahliae isolate 653 exhibited typical verticillium wilt symptoms including wilt, chlorosis and premature senescence, and the effect of V. dahliae was significant for wilt ( 0·0026), AUCPC ( 0·0297) and AUSPC ( 0·0005) in both trials (Fig. 1). The effect of Pectobacterium isolate was significant for lesion size ( 0·0001), wilt incidence ( 0·0001), AUCPC ( 0·0461), AUNPC ( 0·0001) and AUSPC ( 0·0001) in both trials. Wound-inoculations of potato stems with isolate Ec101 alone resulted in typical PED symptoms including wilt, leaf epinasty, chlorosis and premature senescence, while isolate PwO405 caused typical ASR symptoms resulting in rapid and nearly complete necrosis of the inoculated stem (Fig. 1). Stem lesions caused by isolate PwO405 expanded longitudinally along the length of the stem and ranged between 5 and 156 mm long, with limited lateral expansion around the stem. Lesion length ranged from 2 to 12 mm for isolate Ec101. Wounds of water-inoculated plants resulting from the needle injury were small (1–2 mm) and did not expand beyond the initial wound from the needle, indicative of only abiotic injury. Log-transformed lesion length was significantly greater (< 0·0001) in plants inoculated with isolate Ec101 compared to water-inoculated controls and was significantly greater (< 0·0001) in plants inoculated with isolate PwO405 compared to isolate Ec101 or water-inoculated controls (Table 2). Wilt incidence in both trials was significantly greater (< 0·0001) in plants inoculated with isolate Ec101 compared to isolate PwO405. Overall, plants inoculated with isolate Ec101 exhibited greater AUCPC values compared to plants inoculated with isolate PwO405, but AUNPC values were significantly greater in both trials (< 0·0001) when plants were inoculated with isolate PwO405 compared to isolate Ec101 (Table 2). Plant senescence, as indicated by AUSPC values, was significantly greater (< 0·0001) in plants inoculated with isolate PwO405 compared to isolate Ec101 in both trials (Table 2). Lesion size, AUNPC and AUSPC were greater for plants inoculated with isolate PwO405 in the second trial compared to the first trial. Co-inoculations of plants with V. dahliae and either Pectobacterium isolate did not result in greater wilt incidence ( 0·1123), or greater AUCPC ( 0·3421), AUNPC ( 0·1438) or AUSPC ( 0·0731) values compared to inoculations with either pathogen alone, but some differences were observed between trials (Table 2). Disease symptoms were not observed in water-inoculated control plants and the chlorosis and necrosis observed were attributed to natural senescence (Fig. 1).

Table 2. Lesion size (log-transformed), wilt incidence, area under chlorosis progress curves (AUCPC), area under necrosis progress curves (AUNPC), and area under senescence progress curves (AUSPC) in potato plants (cv. Russet Burbank) inoculated with Pectobacterium carotovorum subsp. carotovorum isolate Ec101and Pectobacterium wasabiae isolate PwO405 in the presence and absence of Verticillium dahliae isolate 653a
Pectobacterium V. dahlia b Log(lesion)Wilt incidenceAUCPCAUNPCAUSPC
Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2
  1. a

    Values followed by different letters indicate significant differences among treatments within columns using Tukey's test (= 0·05).

  2. b

    Vdahliae isolate 653: −, absent; +, present.

  3. c

    Significantly different from the Pectobacterium water control and V. dahliae water control treatment combination at = 0·0683.

Water control0·00a0·03a0·00a0·00a253a320b83a174a336a494a
+0·00a0·00a0·81b1·00c540b474bc309a476b849b950b
P. carotovorum subsp. carotovorum isolate Ec1010·73b0·58b0·75b1·00c497abc522c278a335ab775b856b
+0·62b0·61b0·75b0·88c535bc522c414ac453b948bc975b
P. wasabiae isolate PwO4051·71c1·89c0·06a0·19ab299abc14a876b1700c1176c1714c
+1·52c1·91c0·19a0·38b377abc62a807b1605c1184c1667c
Figure 1.

Symptoms observed on potato plants (cv. Russet Burbank) after inoculation with (a) a water-inoculated control representing natural senescence; (b) Pectobacterium carotovorum subsp. carotovorum isolate Ec101 alone; (c) Pectobacterium wasabiae isolate PwO405 alone; (d) Verticillium dahliae isolate 653 alone; (e) isolate Ec101 co-inoculated with isolate 653; and (f) isolate PwO405 co-inoculated with isolate 653.

Quantification of V. dahliae and Pectobacterium in potato stems using quantitative PCR

The effects of V. dahliae and Pectobacterium inoculation on respective Vd(IC) and P(IC) values were significant in both trials when all sample sites were combined (< 0·0001). Significant interactions were not observed between V. dahliae and Pectobacterium treatments on overall Vd(IC) ( 0·2575) or P(IC) ( 0·0755) values using anova in either trial indicating an absence of synergistic, additive or antagonistic effects. Significant differences in overall Vd(IC) values were not observed ( 0·2215) among plants inoculated with V. dahliae alone or together with Pectobacterium in either trial. Nevertheless, greater Vd(IC) values were observed in plants co-inoculated with both V. dahliae and P. carotovorum subsp. carotovorum isolate Ec101 or V. dahliae and P. wasabiae isolate PwO405 compared to plants inoculated with V. dahliae alone in the two trials (Table 3). Co-inoculations of plants with V. dahliae and Pectobacterium did not result in significantly different P(IC) values compared with inoculations with either Pectobacterium isolate alone in either trial ( 0·2089). Significantly greater P(IC) values were observed (< 0·0001) in potato stems following inoculation with isolate PwO405 compared to isolate Ec101 in both trials (Table 4). Pathogen DNA was not detected in water-inoculated control plants in either trial based on melt curve analysis.

Table 3. Verticillium dahliae infection coefficients (Vd(IC)) of V. dahliae isolate 653 quantified in potato (cv. Russet Burbank) stems using real-time quantitative PCR following inoculations with the fungus alone or with one of two Pectobacterium isolatesa
Pectobacterium V. dahliae Vd (IC)
ApicalInoculation siteBasalOverall
Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2
  1. NA, not amplified.

  2. a

    Infection coefficient = cycle thresholdhost/cycle thresholdpathogen. Greater values indicate increased colonization of the host plant tissue by the pathogen. Values followed by different letters indicate significant differences among treatments within columns using Tukey's test (= 0·05).

Water controlWater controlNANANANANANANANA
V. dahliae isolate 6530·724a0·654a0·749a0·725a0·757a0·711a0·744a0·697a
Pcarotovorum subsp. carotovorum isolate Ec101Water controlNANANANANANANANA
V. dahliae isolate 6530·789a0·699a0·856a0·774a0·945ab0·742a0·863a0·739a
P. wasabiae isolate PwO405Water controlNANANANANANANANA
V. dahliae isolate 6530·740a0·610a0·755a0·807a0·983b0·739a0·826a0·719a
Table 4. Pectobacterium infection coefficients (P(IC)) of Pectobacterium isolates quantified in potato (cv. Russet Burbank) stems using real-time quantitative PCR following inoculations with either pathogen alone or with Verticillium dahliae isolate 653a
Pectobacterium V. dahliae P(IC)
ApicalInoculation siteBasalOverall
Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2Trial 1Trial 2
  1. NA, not amplified.

  2. a

    Infection coefficient = cycle thresholdhost/cycle thresholdpathogen. Greater values indicate increased colonization of the host plant tissue by the pathogen. Values followed by different letters indicate significant differences among treatments within columns using Tukey's test (= 0·05).

Water controlWater controlNANANANANANANANA
V. dahliae isolate 653NANANANANANANANA
Pcarotovorum subsp. carotovorum isolate Ec101Water control0·678a0·683a0·732a0·863a0·674a0·709a0·695a0·752a
V. dahliae isolate 6530·711a0·645a0·741a0·922a0·738b0·728a0·730a0·765a
P. wasabiae isolate PwO405Water control1·332b1·298b1·422b1·511b1·371b1·341b1·375b1·383b
V. dahliae isolate 6531·217b1·189b1·342b1·397b1·303b1·408b1·287b1·331b

Significant interactions between Pectobacterium and V. dahliae treatments were also not observed for Vd(IC) values obtained from individual apical ( 0·3079), inoculation site ( 0·3512), or basal ( 0·0685) samples in either trial. However, significantly greater Vd(IC) values were observed in basal samples of plants co-inoculated with P. wasabiae isolate PwO405 and V. dahliae isolate 653 compared to plants inoculated with Vdahliae alone in the first trial (= 0·0412) (Table 3). Generally, Vd(IC) values were greater in samples taken from the basal and inoculation sites compared to apical samples (Table 3). Significant interactions were also not observed for Pc(IC) values in apical ( 0·3079), inoculation site ( 0·3512) or basal ( 0·0685) samples in either trial. The largest P(IC) values were observed in samples taken from the site of inoculation, followed by basal and apical samples for both isolate Ec101 and PwO405 (Table 4). Although not significant, larger P(IC) values were observed in plants co-inoculated with Pcarotovorum subsp. carotovorum isolate Ec101 and Vdahliae isolate 653 compared to plants inoculated with isolate Ec101 alone.

Discussion

Disease complexes can present challenges to plant pathologists as they can be difficult to diagnose, quantify and control (Wallace, 1978). Although it is clear from previous research that individual infection by V. dahliae or Pectobacterium can result in wilt, chlorosis and premature senescence, the role of co-infection by these pathogens on PED was unclear and both additive and synergistic relationships have been demonstrated (Powelson, 1985; Rowe et al., 1987). Previous reports of synergistic or additive interactions varied depending on the disease variables measured, the inoculation method and the Pectobacterium isolates that were used (Kirkland & Powelson, 1981; Kirkland, 1982; Rahimian & Mitchell, 1984b). In addition, the role of co-infection by both pathogens on ASR was not previously studied. The goal of this research was to use traditional greenhouse disease assays, coupled with qPCR, to assess disease severity and quantify pathogen levels in planta following co-infection by Vdahliae and Pectobacterium. Isolates of V. dahliae and Pectobacterium shown to cause specific symptoms when inoculated individually were used for co-inoculations in this study. In general, significant interactions were not detected between Vdahliae isolate 653 and Pectobacterium isolates Ec101 and PwO405 in greenhouse assays regardless of the disease variable measured. In addition, disease variables were not significantly different among plants inoculated with either Pectobacterium isolate alone compared to plants co-infected with V. dahliae. These results indicate that co-infections of potato by Pectobacterium and Vdahliae do not always result in additive or synergistic increases in disease severity. However, the effect of Pectobacterium and V. dahliae co-infection in potato is probably influenced by specific cultivar–isolate interactions and environmental conditions.

Although Rahimian & Mitchell (1984b) observed a synergistic interaction between Pcarotovorum subsp. carotovorum and V. dahliae with regard to disease severity, they determined that populations of the pathogens were not significantly different in plants inoculated with both Pcarotovorum subsp. carotovorum and V. dahliae compared to plants inoculated with either pathogen alone. Similarly, significant increases in pathogen populations were not observed in this study and Pectobacterium and V. dahliae IC values were not significantly different in plants infected with a single pathogen compared to plants co-infected with both pathogens. Significant interactions were also not observed in IC values using qPCR.

Greater Vd(IC) and P(IC) values were observed in plants co-infected with Pcarotovorum subsp. carotovorum isolate Ec101 and Vdahliae isolate 653 compared to plants infected with either pathogen alone. In addition, greater Vd(IC) values were also observed in basal samples of plants co-inoculated with V. dahliae and either Pectobacterium spp. compared to plants inoculated with Vdahliae alone. Although the effect was not additive or statistically significant, these results were consistent between the two trials and are in agreement with a previous study that found increased basal colonization of potato stems by V. dahliae when plants were co-infected by Pectobacterium (Kirkland, 1982). In general, greater Vd(IC) values were observed in the first trial, which may have been a result of the longer period of V. dahliae infection prior to Pectobacterium inoculations (4 weeks compared to 2 weeks). Overall, the results obtained in this study using qPCR are consistent with results found in previous studies which used plate assays and colony counts, although results may differ depending on the types of data collected, potato cultivar, pathogen strains or VCGs, and environmental conditions.

It has been demonstrated that Pectobacterium and other pectolytic bacteria such as Dickeya spp. are capable of systemic movement in potato (Rahimian & Mitchell, 1984a; Czajkowski et al., 2010b). In the present study, the pathogen was detected in both apical and basal portions of aboveground stems, as well as from samples taken from the inoculation point, using both conventional plating (data not shown) and qPCR. Although internal movement of bacteria was detected in both acropetal and basipetal directions, more bacteria were detected in basal stem samples compared to apical stem samples using qPCR, indicating a trend towards basipetal movement of the bacteria in planta. This is consistent with a previous study that also observed an overall basipetal movement of Dickeya chrysanthemi in potato using a strain tagged with a green fluorescent protein (Czajkowski et al., 2010a). However, preliminary experiments using the same wound-inoculation method as this study did not yield pectolytic bacteria from surface-disinfested progeny tubers (data not shown), suggesting that isolates Ec101 and PwO405 did not readily translocate to progeny tubers in a systemic fashion.

Symptoms caused by the two Pectobacterium isolates used in this study differed significantly. Premature senescence, or early dying, caused by Ec101 or V. dahliae alone were nearly indistinguishable. Co-infection by Ec101 and Vdahliae resulted in greater premature senescence than infection with either pathogen alone in both trials, although the difference was not significant. Isolate PwO405 caused similar ASR symptoms in the presence and absence of V. dahliae. A previous study identified differences in stem colonization and stem soft rot severity among Pectobacterium isolates, and isolates of P. carotovorum subsp. carotovorum have been shown to cause stem soft rot (Rahimian & Mitchell, 1984a). Together these results suggest that a significant amount of variation exists among Pectobacterium species and isolates with regards to their abilities to cause ASR and PED. It should be emphasized that these differences are not species-specific, as previous work demonstrated that Pcarotovorum subsp. carotovorum can cause ASR of potato in the greenhouse and in the field (Rahimian & Mitchell, 1984a; Dung et al., 2010) and tuber soft rot capability can vary among and within Pectobacterium species (Marquez-Villavicencio et al., 2011). In this study, isolates Ec101 and PwO405 both caused tuber soft rot in the moderately susceptible cultivar Russet Burbank. However, while isolate Ec101 caused larger lesions on the surface of potato slices in both trials, isolate PwO405 caused deeper lesions and greater tissue loss in both trials, and this difference was significant in one trial. The reason for the differences observed in tissue loss among the two trials is not known, because tubers from the same seed lot were used and the experiments were performed under similar conditions. Nevertheless, both experiments confirmed that both isolates possessed pectolytic ability on potato tuber tissue in spite of the fact that isolate Ec101 did not exhibit pectolytic ability on potato stems. These results indicate that the capability to cause rot in stems and tubers is not correlated in these isolates of Pectobacterium. Previous research concluded that correlations between tuber soft rot and stem rot resistance are rare among potato breeding lines and different mechanisms of host resistance may be involved (Hidalgo & Echandi, 1982). Differences in disease symptoms and severity may be caused by differences in enzyme, toxin, or secreted protein production (Glasner et al., 2008), variations in the ability to colonize and survive epiphytically or in the plant vascular system (Rahimian & Mitchell, 1984a; Elphinstone & Pérombelon, 1986), environmental conditions (Powelson, 1980), differential interactions with host physiology or defence responses (Marquez-Villavicencio et al., 2011), or a combination of factors.

The inoculation procedure used in this study produced consistent results. Differences among replicated blocks were not observed (> 0·05) and results were similar in both trials performed in the greenhouse at different times of the year. The procedure was relatively non-invasive, with water-inoculated plants only exhibiting evidence of abiotic damage consisting of a 1–2 mm wound at the inoculation point and no lesion formation or disease symptom development. In addition, disease symptoms caused by Pcarotovorum subsp. carotovorum isolate Ec101 and P. wasabiae isolate PwO405 were consistently and significantly different, demonstrating that this inoculation procedure can be used to detect differences in pathogenicity and aggressiveness of Pectobacterium isolates on potato stems.

Multiple pathogens are often present in a single potato field and one or several organisms may colonize a particular plant at a given time. Although Vdahliae is often considered to be the primary pathogen involved in PED, pectolytic bacteria may be responsible for early dying symptoms in potato fields from which Vdahliae cannot be detected or in fields not previously cropped to potato (Kirkland, 1982; Kirkland & Powelson, 1982). Field history and environmental conditions can also affect which pathogens play a major role in PED and ASR development (Kirkland, 1982; Kirkland & Powelson, 1982; Powelson, 1985). Correct diagnosis of the fungal and bacterial causal agents of PED is required before the proper control measures can be initiated. An integrated pest management approach which includes sanitation, accurate diagnoses, responsible applications of fumigants and pesticides, sound cultural practices and other available measures can help reduce the impact of early dying and ASR of potato.

Acknowledgements

The authors wish to thank the Washington State Potato Commission for financial support of this project. The authors express special thanks to Drs Tobin Peever and Weidong Chen for critical review of the manuscript prior to submission, and to Jasmin Johnson for excellent technical support. This project was supported by the Department of Plant Pathology (PPNS No. 0594) in the Washington State University College of Agricultural, Human and Natural Resource Sciences, and the Washington State University Agricultural Research Center for CRIS Project No. WNP00652.

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