Evaluation of immunofluorescence with monoclonal antibodies for detecting latent bacterial ring rot infections

Corynebacterium sepedonicum was detected in symptomless potato stems and tubers with immunofluorescence using monoclonal antibodies specific for the bacterial ring rot pathogen. The concentration of bacterial cells in potato tissue preparations ranged from >500 cells/microscope field to 1 cell per preparation. Symptomless tubers containing ring rot bacteria planted in field plots yielded plants with ring rot symptoms, plants with latent ring rot infections, or plants with no detectable levels of ring rot bacteria. Tubers with the greatest number of bacteria were most likely to develop plants expressing ring rot symptoms, but even some seed tubers with a low number of bacteria developed into plants with symptoms. Some seed tubers with high levels of ring rot bacteria produced plants with only low numbers ofC. sepedonicum.     

Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

Resistance of tubers from different potato cultivars to soft rot caused byErwinia carotovora subsp.atroseptica

Mechanically harvested tubers of 14 potato cultivars grown on both loamy sand and silt loam soils were evaluated for resistance to bacterial soft rot caused byErwinia carotovora subsp.atroseptica. Cultivars were also assayed for calcium and dry matter content to determine possible correlations with soft rot resistance. Resistance of potato tubers to bacterial soft rot was assayed after harvest by inoculating tubers and determining percent surface area decayed after four days in a mist chamber. Tubers of the cultivars differed widely in resistance to bacterial soft rot. The ranking of the resistance of tubers from different cultivars to bacterial soft rot was similar for both soils, although disease severity in tubers from a particular soil type varied with the season. Of the cultivars tested, tubers of Butte, Russet Burbank, and Nor-gold Russet were ranked as the most resistant, whereas tubers of Redsen, Norchip, Norland and Belchip were rated as the most susceptible. Tubers of Rhinered, Red La Soda, Superior, Pontiac, Atlantic, and Nooksack were intermediate in resistance. Neither calcium concentration in medullar tissues nor dry matter of potato tubers alone could be correlated with ranking of the cultivars with respect to severity of bacterial soft rot. However, a multiple linear regression model incorporating both variables indicated that tubers with a high content of both calcium and dry matter were likely to be less susceptible to bacterial soft rot than were tubers with lower percentages of these variables.     

Suppression of bacterial soft rot in potato tubers by application of kasugamycin

An immersion in aqueous solutions of Kasumin®, containing kasugamycin at 10 to 320 mg/L (ppm) for 1 to 320 sec delayed or prevented the development of soft rot on inoculated (Erwinia carotovora subsp.carotovora) tuber disks incubated over wet filter paper at 24C for 24 to 72 hr. Treatment efficacy decreased as inoculum level and incubation period increased. Short immersion periods were not as effective as longer ones. When stored tubers were inoculated withE. c. carotovora and incubated up to 5 days in fog chamber at 20 C, immersion treatments in 20 to 400 ppm kasugamycin either had no effect or increased soft rot development. By contrast, when the stored tubers were cut into sections before inoculation and treatment, immersion in 300 ppm reduced soft rot development on the cut surfaces from 83% (inoculated controls) to 3.2%. With non-inoculated sections, treatment with 25 ppm reduced the SAD from 13.3 to 0.6%. Wash or rinse treatment of freshly harvested tubers in 40 to 160 ppm produced a significant reduction in the severity of bacterial soft rot among tubers incubated for 72 to 96-hr. The 160-ppm treatment delayed disease onset by at least 24 hr. Pre-washing freshly harvested tubers with clean or chlorinated water prior to antibiotic treatment did not consistently improve the efficacy of the kasugamycin treatment.     

Comparison of the effectiveness of different methods of screening for bacterial soft rot resistance of potato tubers

Tubers from eight potato cultivars (cvs) grown at two different locations in Wisconsin were tested for bacterial soft rot resistance using different inoculation techniques. The procedures included 1) point inoculations of tubers with different inoculum levels followed by incubation in ambient or low oxygen condtions, 2) inoculation of mechanically bruised tubers followed by incubation in a mist chamber, and 3) a standard slice inoculation method. The point titration test followed by incubation in dew chamber and the mist chamber-bruise test showed similar patterns of resistance for cultivars that were used in these experiments. These two methods are considered to be effective for screening potato tubers for bacterial soft rot resistance. Point titration methods are very useful if only limited numbers of tubers are available. The mist chamber-bruise test is simpler than the other procedures; however, to obtain reproducible results large numbers of tubers are required. Because of the great variability of the results obtained in inoculation of slices, the reliability of this approach can be questioned as a standardized method for evaluation of resistance. Tubers of somatic hybrids ofS. brevidens andS. tuberosum and their sexual progeny were significantly more resistant to bacterial soft rot than tubers of moderately resistant cultivars when evaluated by each of the assay procedures.     

The role of stem inoculum in the transmission ofFusarium sulphureum to potato tubers

Summary Over 2 years, seed tubers or stems were inoculated withFusarium sulphureum Schlecht. (F. sambucinum Fuckel f. 6 Wollenw.) and the incidence of dry rot on daughter tubers assessed by standardised wound tests. The amount of stem and soil inoculum was also measured in one year. Inoculating stem bases resulted in greater concentrations of spores on the stem bases, greater soil populations at harvest and more dry rot on tubers than inoculating the seed tuber. Overall, the incidence of dry rot and the amount of stem and soil inoculum was similar for the three methods of haulm destruction: cutting and removing the stems, applying sulphuric acid or applying diquat dibromide to the growing plants.     

The incidence of decay and factors affecting bacterial soft rot of potatoes

The incidence of tuber decay in rail car shipments of potatoes from the State of Washington was determined from 1965 to 1969 inclusive. Reinspection certificates revealed that 7.9% of the rail cars of potatoes shipped during this period were rejected due to tuber defects; tuber decay accounted for 63% of these rejected cars. Percentage of rejections due to tuber decay was greater for Russet Burbank than for Norgold Russet potatoes. Temperature, moisture, and oxygen levels influenced development of bacterial soft rot in freshly harvested potatoes and in potatoes stored for 3 months at 35 F (2 C) previous to treatment. When tuber surfaces were kept dry, no soft rot developed in 11 days over ranges of 50–70 F (10–21 C) and 2–20.5% oxygen levels. Freshly harvested tubers partially immersed in water did not rot when cooled to 40 F (4.4 C) in < 24 hours and held for 8 days under either 20.5 or 4% oxygen levels; however, tubers, rotted when stored similarly but at 50 F or higher temperatures. Partially immersed tubers incubated for 1 day or longer at 70 F developed soft rot in a low oxygen (4%) when later stored for, 8 days at 40 F. When rot occurred, the amount was always greater under lower oxygen levels irrespective of storage temperature. Of various cooling methods tested in rail cars loaded with 100 lb (45.4 kg) sacks of potatoes, prechilling of tubers and icing of cars prior to loading lowered floor temperatures most rapidly. Icing cars after loading lowered floor temperatures the slowest. In a carton shipment, the temperature remained above 68 F (20 C) in cartons after 8 days in transit, even though the wall and floor temperatures had cooled to near 55 F (12.8 C) by the second day. Oxygen level surrounding, tubers in the center of car loads of 100 lb sacks of potatoes was approximately 20.5% after 8 days transit.     

Bacterial soft rot potential in washed potato tubers in relation to temperatures of tubers and water during simulated commercial handling practices

The bacterial soft rot potential in potato tubers was affected by differences in temperature between tubers and suspensions ofErwinia carotovora at the time of inoculation by immersion. In general, disease severity after incubation was higher when tuber temperatures were higher as opposed to equal to or lower than that of water containing cells of the bacterium. Disease severity was also greater when tubers were stored at 20° to 23°C in contrast to 4° or 26° to 30°C for 16–18 h prior to immersion. Tubers with a pulp temperature of 8°C were more prone to bruise injury as compared with those at 18°C when struck with a pendulum type bruising instrument. When the injured tubers were inoculated and incubated, most bruises became infected with soft rot bacteria.     

Potato sprout inhibition and tuber quality after treatment with high-energy ionizing radiation

The use of industrial-type linear accelerators may be an alternative treatment for controlling sprouts in potatoes during long-term storage. The technical aspect of treatment has been described and may include largescale treatment of bulk potatoes as they are moving along a system of conveyors either to or from storage. The objective of the study was to evaluate the use of a linear accelerator for long-term sprout control under commercial storage conditions and the effects on tuber quality. An 18-MeV linear accelerator was used to provide sprout-inhibiting irradiation to various lots of ‘Russet Burbank’ potatoes. Successful sprout suppression was achieved with doses of 40 to 50 Gy (0 g sprout weight after 9 months of storage at 7.2 C) while higher doses caused undesirable increases in reducing sugars in the tubers. Glucose concentrations of treated tubers (0.25% fresh tuber weight) were higher after treatment than the untreated controls (0.08%), but returned to control levels after 2 to 6 months in storage. Glucose concentrations in treated tubers were significantly reduced with storage management for reconditioning by increasing the storage temperature for one month (from 7.2 C to 14.4 C) resulting in glucose concentrations near control concentrations. By increasing the dose-rate from the accelerator, recovery of tuber glucose concentration due to radiation treatments was significantly faster than when the dose was given more slowly. Potato tubers irradiated with high rates (100 Gy) of high-energy electrons exhibited an increase in soft rot and dry rot incidence in storage. Disease development in potatoes treated with sprout inhibiting doses of radiation (50 Gy) increased in lots with a high rot potential. Treated lots with low rot potential were successfully stored for 8 months or more without an increase in rot potential or shrinkage. Selection of lots for irradiation treatment should include some preliminary evaluation for development of disease in long-term storage.     

Fungicidal control ofPhytophthora erythroseptica: The cause of pink rot on potato

Two shade house and six field experiments were undertaken to evaluate fungicides and timing of application for the control of pink rot of potatoes caused byPhytophthora erythroseptica. Pink rot developed in up to 60% of tubers grown in artificially inoculated soil and 21% of the tubers in naturally infected soil. Ridomil (metalaxyl) and Ridomil Gold (mefenoxan) granules applied to the soil at planting completely or nearly completely controlled pink rot throughout the season in both pot and field experiments. Foliar applications of Ridomil Gold MZ (mefenoxan plus mancozeb) applied when tubers were around 20 mm diameter and another one or two sprays 12 to 28 days later also provided more than 80% control of pink rot in most experiments. Foliar applications of Shirlan (fluazanim) or Acrobat MZ (dimethomorph plus mancozeb) showed some activity against pink rot, but were significantly less effective than Ridomil Gold MZ. Foli-R-Fos 200 (phosphonic acid) or Dithane M45 (mancozeb) applied to the canopy showed little activity against pink rot in most experiments. Yield increases of between 6 and 17 tons ha^?1 resulted from the application of either Ridomil granules at planting or two to three Ridomil Gold MZ foliar sprays around “tuber set.”     

The effect of potato virus X on susceptibility of potato tubers toFusarium roseum ‘Avenaceum’

Potato virus X (PVX)-free and mild PVX-infected tuber lines originating from the same Katahdin clone were compared for susceptibility to tuber rot incited byFusarium roseum (Lk.) Synd. and Hans. ‘Avenaceum’. The effect of interval between top-kill and harvest on development ofFusarium tuber rot was also determined. A series of tubers from both PVX-free and PVX-infected tuber lines was either (i) mechanically bruised and inoculated by immersion in a spore suspension ofF. roseum ‘Avenaceum’, (ii) mechanically bruised but not inoculated, (iii) inoculated only or (iv) neither bruised nor inoculated. Tubers from all treatments were then stored at temperatures of 18–21 C (65–70 F) for a 3-week period prior to being placed at 4 C (39 F) for the remainder of the storage period. The amount of rot was determined after a 5–6 month storage period at the latter temperature. In a 4-year study PVX-free tubers were significantly (1% level) more susceptible to infection byF. roseum ‘Avenaceum’ than tubers infected with mild PVX. PVX-free tubers left in the ground for 2 or 3 weeks after top-kill were significantly (1% level) more susceptible toFusarium dry rot than those infected with the virus; however, there was no significant difference in disease incidence between PVX-free and PVX-infected tubers when harvested 4–5 weeks after top-kill. Tubers, both PVX-free and PVX-infected, remaining in the ground for a 2 week period following top-kill were significantly (1% level) more susceptible toFusarium tuber rot than those harvested 3 weeks after top-kill; similarly, both PVX-free and PVX-infected tubers harvested 3 weeks after top-kill were significantly (1% level) more susceptible than those left in the ground for a 4–5 week interval.     

Detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers grown from seed pieces with various levels of inoculum

Summary An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was used to monitor potato stems of three cultivars for the presence of the ring rot pathogen,Clavibacter michiganensis subsp.sepedonicus, during the growing season and in progeny tubers after harvest. The highest ELISA values were obtained with the highest concentration of bacteria used to inoculate seed pieces in all cultivars tested. Low ELISA values were obtained for stems and progeny tubers selected from plants grown from seed inoculated at lower bacterial concentrations. Estimates of bacterial densities in stems and progeny tubers by immunofluorescence indicated that low ELISA values were most probably caused by low bacterial numbers. It is suggested that the sensitivity of ELISA for detecting the ring rot pathogen in potato stems and progeny tubers is a function of the concentration of bacteria in individual seed pieces.     

Reducing the potential for bacterial soft rot in potato tubers by chemical treatments and drying

The potential for bacterial soft rot caused byErwinia carotovora in freshly inoculated potato tubers was reduced up to 99% by immersion for 5 min in solutions of sodium hypochlorite (chlorine bleach) containing up to 10,000 ppm chlorine. Reductions up to 93% were achieved using a combination treatment of immersion in 1% citric acid for 5 min followed by airdrying. Immersion treatment alone in 1% aqueous solutions of citric, acetic, ascorbic, or malonic acid also significantly reduced the soft rot potential. In contrast, no reductions accompanied treatment with solutions of potassium or calcium acetate. All treatments including the combination immersion/airdrying treatment were much less effective if tubers had been infiltrated initially with soft rotErwinia or had numerous mechanical injuries. If tubers had not been infiltrated with the causal organism, immersing them in 1% citric acid reduced the potential nearly as much as a similar treatment with 1000 ppm chlorine. A 30-sec immersion in the latter was less effective than a similar treatment with 500 ppm a.i. CGA 78039, an experimental bactericide. However, air-drying, coupled with provisions for keeping tuber surfaces free from moisture, remains the most effective means of reducing losses to bacterial soft rot.     

Determination of the potential for development of bacterial soft rot of potatoes

A method has been devised for assessing the potential hazard of soft rot development in potatoes. Tubers are placed in a mist chamber at 20 C (68F), a film of water is maintained on the tuber, and the extent of decay is assessed after 5 days. The soft rot potential determined in this way is intended as apractical indication of potential storage hazard, reflecting the overall effect of the various factors leading to bacterial decay. Tubers that had passed through a flume system and a rinsing spray in a commercial packing plant had a greater decay potential, even after passage through a hot-air dryer, than unwashed tubers. Differences in decay potential could be demonstrated between tubers that had been mechanically harvested and loaded into storage bins and those that had been harvested manually. In addition to the effect of mechanical damage and of passage through flumes, the decay potential was probably affected by the inoculum level ofErwinia carotovora. Pectolytic clostidia were also usually present in tubers rotting in mist chambers, and are probably involved in the decay.     

应用PCR技术快速检测马铃薯环腐病菌

根据马铃薯环腐病菌16SrDNA基因片段核苷酸序列设计引物(引物1∶5-′TGTACTCGGCCAT-GACGTTGG-3′和引物2∶5-′TACTGGGTCATGTTGGT-3)′,进行马铃薯环腐病菌PCR特异性扩增试验。合成的引物能从马铃薯环腐病菌总基因组DNA和细菌纯培养,以及人工接种和自然发病的马铃薯块茎中特异性扩增环腐病菌16SrDNA基因片段1046bp。该试验结果为马铃薯环腐病的鉴定、检测及病害流行学研究提供了新的技术和方法。     

Inoculation of potato tubers withErwinia carotovora during simulated commercial washing and fluming practices

The potential for bacterial soft rot in potato tubers was affected by laboratory simulation of some components of fluming and washing practices at commercial packinghouses. The potential was expressed as disease severity (average percentage surface decayed) after a standard 4-day incubation at 20°C in a mist chamber. Increased disease resulted when there was an increase in the following factors: a) duration of immersion of tubers in water; b) population of bacteria suspended in water; c) hydrostatic pressure on submerged tubers, and d) concentration of a surfactant (Triton X-100) in the suspensions. A sample of tubers from each tuber lot was uniformly moistened with tap water and then incubated to determine a base-level disease severity. Disease severity in Russet Burbank tubers from commercial storage increased from the base-level of 0.8% to 76% when tubers were immersed in 5 × 10⁶ colony-forming units (cfu)/ml ofErwinia carotovora pv.carotovora (Ecc) for 64 min prior to incubation. Disease severity was 64% when tubers were immersed in a similar suspension for only 0.1 min, but with a hydrostatic pressure of 230 cm of water. Severity values fourto fivefold above control levels (from immersion in water) resulted from an immersion for 5 min in 5 × 10³ cfu/ml with hydrostatic pressures of 5, 180, or 370 cm of water. A 20-fold increase in severity occurred when Red LaSoda tubers were immersed briefly (less than 5 s) in 5 × 10⁶ cfu/ml. Rinsing these tubers with tap water after inoculation, but prior to incubation, did not affect the subsequent development of decay. Thus, the high potential for bacterial soft rot in tubers that have been flumed or washed by water concentrated with soft rotErwinia cannot be reduced by washes or rinses with clean water.     

马铃薯软腐病菌的16SrDNA PCR检测

马铃薯软腐病是威胁马铃薯块茎的细菌性病害之一,为了提高马铃薯的品质与产量,减少贮藏期马铃薯块茎的腐烂,对马铃薯进行软腐病检测与鉴定是十分必要的。利用16SrDNA PCR对马铃薯软腐病原菌的进行检测,证实16SrDNA PCR检测技术是可行可靠的。在对PCR引物的筛选中,要根据所检测的对象进行有效、合理的设计,才能达到快速有效的目的。以马铃薯软腐病菌为代表,利用16SrDNA PCR进行鉴定,达到了预期的目的,为病原菌的快速鉴定方法提供了依据。     

A rapid scoring technique for potato tuber disease assessment

Summary Three methods of scoring disease severity were compared on potato tubers infected with either common scab, gangrene or dry rot. For each disease, replicated samples of infected tubers were taken of 12–14 cultivars representing a range of reaction from resistant to susceptible, and disease severity assessed by 1) estimating the proportion of the surface area affected by the disease, 2) calculating the percentage of tubers infected to a pre-determined amount, 3) an overall ‘glance score’ on a 1–9 scale of decreasing infection. The third method gave scores which correlated closely with the others, and since it is direct and simple to use, it has become the preferred method in disease screening tests at the Scottish Crop Research Institute.     

First report ofPythium sylvaticum causing potato tuber rot

Leak is a post-harvest disease of potato tubers manifested by a wet, spongy rot that can quickly lead to complete tuber decay. In North America, leak is most commonly caused byPythium ultimum var.ultimum Trow, although a number of otherPythium spp. have been found to be pathogenic to potato tubers. We report that an isolate identified by DNA sequencing asP. sylvaticum Campbell & Hendrix was pathogenic to potato tubers. The organism produced a dark, hollow cavity in tubers that was much drier in texture than typical symptoms of leak. Symptomology was similar in tubers of the cultivars Yukon Gold, Green Mountain, and Russet Burbank. Studies to complete Koch’s postulates confirmed thatP. sylvaticum could be reisolated from diseased tissue and produced the same disease symptoms when isolates were subsequently re-inoculated into healthy tubers. This is the first report ofP. sylvaticum causing rot in potato tubers.     

Influence of temperature and length of storage on resistance of potato to tuber rot induced byErwinia chrysanthemi

Tubers of 11 clones ofSolanum tuberosum subsp.andigena (andigena) and 12 cultivars ofS. tuberosum subsp.tuberosum (tuberosum) were inoculated with water suspensions (5.5 × 10⁴ 5.5 × 10⁵, 5.5 × 10⁶ CFU/ml) ofErwinia chrysanthemi. Tubers were inoculated immediately after harvest and after 6 and 16 wk of storage at 4 and 23°C. Tuber rot incidence in andigena and tuberosum increased as inoculum concentration increased. Based on tuber rot severity, clones of andigena were classified as resistant, intermediate, or susceptible and cultivars of tuberosum were classified as intermediate or susceptible. Rot severity increased in all tubers stored at 4°C and in tubers of tuberosum stored at 23°C; rot did not increase in tubers of andigena stored at 23°C. Electrolyte leakage (EL), total sugars (TS), reducing sugars (RS), non-reducing sugars (NRS) and dry matter (DM) were determined in non-inoculated tubers. There was a significant positive correlation between the rate of EL, concentration of RS and tuber rot in andigena and tuberosum. Tuber rot and DM were negatively correlated in tubers of andigena; but they were not correlated in tuberosum. Clones of andigena with low EL, TS, RS, and high DM were resistant to tuber rot, and the incidence of tuber rot in these clones was much less influenced by temperature and length of storage. The influence of temperature and length of storage on susceptibility toE. chrysanthemi may be explained by increased cell membrane permeability; increased leakage of accumulated sugars in potatoes stored at 4°C could favor bacterial proliferation resulting in more disease.