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Characterization of the causal organism of blackleg and soft rot of potato, and
management of the disease with balanced fertilization

Article in Pakistan Journal of Botany · December 2014

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Pak. J. Bot., 46(6): 2277-2284, 2014.

CHARACTERIZATION OF THE CAUSAL ORGANISM OF BLACKLEG AND

SOFT ROT OF POTATO, AND MANAGEMENT OF THE DISEASE
WITH BALANCED FERTILIZATION
HAFIZ FARHAD ALI1*, AYESHA BIBI1, MUSHARAF AHMAD1, MUHAMMAD JUNAID1,
ASAD ALI1, SHAUKAT HUSSAIN1, SHAH ALAM2 AND SARTAJ ALAM1
1
Department of Plant Pathology, 2Department of Plant Protection. The University of Agriculture, Peshawar.
*
Corresponding author e-mail: hafizfarhad66@yahoo.com
Abstract

Based upon colony morphology, physio-biochemical tests and polymerase chain reaction (using species or subspecies-
specific primers) studies, 20 isolates (out of a total of 42) were found to be Erwinia carotovora subspecies atroseptica (Eca),
19 were identified as Erwinia carotovora subspecies carotovora (Ecc), and 3 as Erwinia chrysanthemi (Ech). Results of
the subspecies-differentiating biochemical tests indicated that majority of the candidate Ecc isolates did not produce acid
from α-methyle glucoside (as expected) but their reaction to the production of reducing substances from sucrose was
variable. Likewise, some of our Eca and Ecc strains (unexpectedly) were sensitive to erythromycin. Also, most of our Eca
strains unexpectedly grew at 36oC. Our strains slightly deviate from the standard description in some of their minor
characteristics but they still remain the valid members of the Eca, Ecc or Ech group as similar variations in minor
characteristics have been found by other workers. The occurrence of intermediate forms of Eca and Ecc (sharing some of the
characteristics of both the groups) indicates variability happening among these strains. This variability indicates the potential
ability of the pathogen to break the resistance of the host. The results of the effect of balanced nutrition in controlling
blackleg and soft rot of potatoes indicated that the fertilizer combination of N3P1K3 (262/252/262 kg.ha-1) which is slightly
higher than the normally practiced dose (247/247/247 kg.ha-1) was the best in bringing the disease to a minimum and
subsequently increasing the yield.

Key words: Erwinia carotovora subsp. atroseptica, Eca, Blackleg, PCR, isolates.

Introduction rotting of potato in temperate climates (Pitman et al.,

2010). Pathogenicity of Eca is usually restricted to
Potato (Solanum tuberosum L.) is one of the most potatoes grown in cool and temperate climates, while
important vegetable crops of the world including Ecc (causing potato soft rot and, in some cases blackleg
Pakistan. The climate of Pakistan, especially that of too) have a wider distribution in both temperate and
Khyber Pukhtunkhwa, is very suitable for the production tropical zones showing wider host ranges than those of
of potatoes. In Pakistan, potato occupies an area of the other subspecies (Wells & Moline, 1991). Eca is the
133435 ha with a total production of 2581554 tons, while major cause of blackleg, a blackening of the stem base
in Khyber Pukhtunkhwa, it occupies an area of 9600 ha of potato plants, which originates from the mother tuber
with a total production of 129529 tons (Anon., 2006- (Pérombelon & Kelman, 1987). Black leg causes 10-
2007). Among the 24 districts of Khyber Pukhtunkhwa, 30% whereas soft rot causes 2-10% losses in Khyber
Nowshera, Mardan, Abbotabad, Swat, Dir Upper, Chitral, Pukhtunkhwa (Turkensteen, 1986). The pathogens are
and North Waziristan are the major potato-growing seed-borne and frequently remain undetected by the
districts where during 2006-2007, area under the crop was common detection methods. As much as 20% of the
1049, 868, 550, 917, 976, 759 and 890 ha with an average seed potato supplied (from open market of Punjab) on
production of 17334, 7518, 5888, 16257, 20737, 11385 credit to small potato farmers in Khyber Pukhtunkhwa
and 9075 tons respectively (Anonymous, 2006-2007). The by the potato seed dealers is infected with Ecc & Eca
average per ha production of potatoes in Khyber (Musharaf Ahmad, Professor, Department of Plant
Pukhtunkhwa is well below its potential. One of the Pathology, The University of Agriculture, Peshawar,
reasons for this low yield and quality is the occurrence of Personal Communication).
different bacterial diseases such as brown rot, ring rot, Since potato seed tubers are the chief source of
black leg and soft rot of potatoes. inoculum for many bacterial diseases, planting of
Blackleg and soft rot are important diseases (caused pathogen-free seed tubers is a must for the control of such
by Erwinia spp., a Gram-negative, facultatively diseases. However, the traditional methods for the
anaerobic, rod-shaped bacterium with peritrichouse identification and detection of these bacterial pathogens in
flagella) of potato that cause heavy losses to potato crop seed tubers are quite laborious and not really fool-proof.
not only in the field but also in the storage where the Polymerase chain reaction (PCR) that rapidly detects,
bacteria are transmitted from diseased tubers to healthy identifies and characterizes microorganisms in a shorter
ones. Multiple subsp. of Erwinia, including E. time is a good alternate for the identification purposes.
carotovora subsp. atroseptica (Eca) and E. c. subsp. Gene-specific/ subspecies-specific/ species-specific primers
carotovora (Ecc), E. chrysanthemi (Ech) E. carotovorum could be used in a simple or multi-plex PCR to precisely
subsp. brasiliensis (Ecb), and Pectobacterium (syn. identify the seed-borne phyto-pathogenic bacteria. Based
Erwinia) wasabiae (Pw) attack potatoes. Ecc and Eca upon PCR-indexing results, seed tubers could be treated to
are the primary enterobacteria responsible for soft free them of the pathogens.
2278 HAFIZ FARHAD ALI ET AL.,

Controlling plant bacterial pathogens and the diseases were put in paper bags, kept cool and processed as soon
they cause is a very serious problem. In case of human and as possible (to reduce the chances of secondary invaders)
animal bacterial diseases, antibiotics are frequently used for in The University of Agriculture Clinical Plant Pathology
the control of such diseases. However, to control Laboratory. Samples yielding bacterial colonies having
phytobacterial diseases, antibiotics are not recommended the morphology of Soft Rot Erwinias (SRES) were
for two reasons; first, antibiotics are expensive and second, recorded whereas those yielding no bacteria or G+
their long-term use might force bacteria to develop bacteria were discarded.
resistance against these antibiotics. This antibiotic
resistance, when transferred to animal and human bacteria, Isolation of Erwinia spp.: Nutrient agar or NA (Bacto
can pose serious threats. Use of copper fungicides as Agar; 10gm, NaCl; 5.0 gm, K2HPO4; 5 gm, KH2PO4;
bactericides is another option to control some plant 2gm, Bactopeptone; 1.0gm and Ditilled water 1L) was
bacterial diseases to some extent. However, copper used for the isolation of soft rot Erwinias. Tubers and
compounds, besides being bad for the environment, can plant samples showing disease symptoms were cleaned,
cause phytotoxicity and may exert a negative impact on the surface-sterilized with 0.5% sodium hypochlorite solution
yield (Kowalska & Smolinska, 2008). (for 30 seconds), washed with sterile distilled water, and
Proper fertilization, both the amount and the type of ground in sterile 0.85% saline solution using sterile
fertilizer could be manipulated to control some plant mortar under aseptic conditions. The resulting bacterial
bacterial diseases. Nitrogen content of chicory plants was suspension was left undisturbed for a few minutes. A
found to be positively correlated with the amount of loopful of this suspension was then streaked on the
nitrogen fertilization (Reerink, 1993) and with the amount surface of plates containing nutrient agar, and the plates
of the bacterial soft rot (Wright, 1993). Likewise, Brigitte were incubated at 28oC for 24 h. Individual colonies
et al., 1999) concluded that nitrogen treatment enhanced (transparent, circular, raised, shiny and creamy white)
soft rot incidence in chicory heads. McGovern et al., growing on NA were picked up, re-suspended in 0.85%
(1985) compared three different types of N fertilizers saline and streaked on NA plates, and then incubated at
while studying the susceptibility of Chrysanthemum 28oC for another 24 h. This was done several times to
morifolium to Erwinia chrysanthemi. They found that Ca obtain pure cultures.
(NO3)2 and NH4NO3, at 400 ppm level, were better than Nutrient agar is a non-selective medium and
(NH4)2SO4 in terms of reducing the disease susceptibility sometimes there is a problem of over-growth of
of Chrysanthemum morifolium. Some fertilizers can also saprophytic bacteria on this medium. Therefore, in some
increase or decrease the level of pathogeicity of a cases, we used an indirect approach to avoid this problem.
pathogen. For example, Gracia et al., (2004) For this purpose, green pepper fruits were used as an
experimentally proved that one of the reasons of the enrichment host for the soft rot Erwinias which were
increased tuber rot was the activation of bacterial tissue subsequently isolated on NA. The peppers were surface-
macerating pectic enzymes by phosphorous of the growth disinfested with 70% alcohol and 1% sodium hypochlorite
medium suggesting that P fertilizers could increase plant (NaOCl), for 30 seconds each, and then washed with sterile
host susceptibility to soft rot bacteria. Potassium also distilled water. Next, sterile toothpicks were stabbed into
plays an important role in defending plants against
soft-rotten tubers or the margin of blackleg lesions on
diseases including bacterial diseases. It was found that the
potato stems and then the same toothpicks were inserted
use of potassium decreased the incidence of 70% of
into green pepper (Capsicum annuum L.) fruits (Takatsu et
fungal diseases, 69% of bacterial disease, and 41% of
viral disease. Simultaneously, K increased the yield of al., 1981). The inoculated fruits were kept in a humid
plants infected with fungal disease by 42%, with bacterial chamber at 28oC for 24-48 h. Decayed tissue was peeled
disease by 57%, and with viruses by 78% (Perrenoud, off with a scalpel and crushed in 0.85% saline as described
1990). Potassium enables plants to produce disease before. A loopful was used to streak the surface of NA
inhibitory compounds, such as phenols and phytoalexins. plates. Single colonies were harvested and purified as
If K is low in plants, inorganic N would accumulate described before.
which results in the rapid break down of phenols thus Pure colonies were saved in 70% glycerol solution
making plants susceptible to diseases (Kiraly, 1976). and stored at -20oC or -80oC. Cultures were also saved in
Keeping in view the importance of potato crop in 0.85% sterile saline solution and stored at 4oC. When
Khyber Pakhtunkhwa, the losses caused by blackleg and needed, each bacterial strain was cultured on LB (Trypton
soft rot diseases to this crop, and the lack of research 10gm, yeast extract 5gm, NaCl 10 gm, agar 15.0 gm,
work on these diseases in this province, the present distilled water 1 liter) at 28°C for 2 days.
research work was carried out to characterize and PCR-
identify the pathogen(s), and to explore the role of proper Identification of bacteria: Identification of the causal
fertilization of potato plants in controling blackleg and organism as Eca, Ecc or Ech was done by colony
soft rot of potato. morphology, physio-biochemical tests (such as tissue
maceration, yellow pigmentation on YDC, growth at
Materials and Methods 36oC, mucoid growth, reducing substances from sucrose,
5% NaCl tolerance, erythromycin sensitivity, catalase
Sample collection: A large number of potato plants and activity, and acid production from alph-methyle
tubers showing typical symptoms of blackleg and soft rot glucoside) and polymerase chain reaction (PCR). Tissue
were collected to isolate the causal organism. Samples maceration test was carried out as reported by El-
CHARACTERIZATION OF THE CAUSAL ORGANISM OF BLACKLEG ROT OF POTATO 2279

Hendawy et al., (2002) and the physio-biochemical tests kept at room temperature for a few days in the laboratory
were performed according to the methods reported by for sprouting. Data were taken on disease incidence,
Lelliot & Dickey (1984). severity, and yield 70 days after sowing.

Molecular identification: The identity of the bacteria was Disease incidence and severity: Disease incidence (%)
confirmed by polymerase chain reaction (PCR) using was simply calculated by dividing the number of plants
subspecies-specific primers. Eca-specific primers, Eca1F (per each random spot of 1 M2) showing blackleg and soft
(5'-CGGCATCAT-AAAAACACG-3') and Eca2R (5'- rot disease symptoms by total number of plants and
GCACACTTCATCCAGCGA-3') (De Boer & Ward, multiplied by one hundred. The values of all random spots
1995) amplified, as expected, a 690 bp band. Ecc-specific (per field) were averaged together. Disease severity of
primers, EXPCCR (5’-GCCGTAATTGCCTACCTGCTT- each plant present in each randomly selected spot was
AAG-3’) and EXPCCF (5’-GAACTTCGCA- assessed on a scale of 0-3 as reported by Wright et al.,
CCGCCGACCTTCTA-3’) (Kang et al., 2003) produced a (2005) where 0 = no disease symptoms on plant, 1 = ≤
550 bp band whereas Ech-specific primers, ADE1 (5’- 50% of the plant has disease symptoms, 2 = > 50 % of the
ATCAGAAAG-CCCGCAGCCAGAT-3’) and ADE2 (5’- plant has disease symptoms, and 3 = plant totally dead.
CTGTGGCCGA- TCAGGATGGTTTTGT-CGTGC-3’) The disease rating values of individual plants were
(Nassar et al., 1996) amplified a small band of 420 bp size. converted to % disease severity values using Bdliya &
DNA was extracted (Wang et al., 1993) from the unknown Dahiru, (2006) statistical equation (S = 100∑n/3N; where
(target) bacterium and 3 µl of the lysate (template) was S = black leg severity (%), ∑n = sum of the ratings of all
directly used in PCR reaction. The PCR master mix plants, N = shows the number of plants used for rating,
included 2 mMol l−1MgCl2, 1 µMol l−1 each primer, and 0.2 and 3 = biggest number of the disease rating scale) to
mMol l−1 dNTPs. The concentration of the Taq buffer (Tris determine % disease severity. Values of all spots per field
HCl pH 8.8) used was 67mM l−1. To make the Taq DNA were averaged.
polymerase work for longer time, the enzyme was added to
PCR tubes after the initial denaturation step. MJ mini Results and Discussion
thermocycler (Bio-rad, USA) PCR machine was used to
amplify DNA. PCR steps and temperatures used were (i) Identification of soft-rot Erwinia spp. (SRES): All the
one-time denaturation at 95oC for 5 minutes, (ii) repeat isolates either obtained from soft rotted tubers or black-
cycle denaturation at 94oC for 30 sec, (iii) primer annealing legged potato plants were identified by colony
at 47o C for 30 sec, and (iv) primer extension at 72oC for 50 morphology and physio-bicchemical tests. Regarding the
sec. The denaturation, primer annealing and primer morphology of the bacterial colonies on nutrient agar
extension steps were repeated 39 times. At the end of 40 (NA), the colonies appeared to be transparent, circular,
cycles, a final extension at 72°C for 8 min was done. raised, shiny and creamy white after 48 h incubation at
However, the temperatures used for primer annealing steps 28oC (Fig. 1). All the strains were catalase-positive,
in case of PCR-identification of Ecc and Ech were 57oC tolerated 5% NaCl, macerated potato (Fig. 2) and carrot
each. After the amplification of the DNA through PCR, 25 tissue, and produced yellow pigment on YDC (Table 1).
µl from each sample tube (PCR tube) was taken and The above-mentioned tests are general tests and do
electrophoresed through a 2% (w/v) agarose gel (Sambrook not differentiate Eca from Ecc or Ech. In order to know
et al., 1989) to separte the amplified DNA bands. The whether our isolates belong to Eca, Ecc or Ech, key
separated bands were stained with ethedium bromide diagnostic tests such as acid production from α-methyle
(0.5µg/ml) solution on agarose gel, visualized under UV glucoside, production of reducing substances from
light in UV tech machine (ESSENTIAL, D-55-20-M-Auto, sucrose, sensitivity to erythromycin and growth at 36oC
UK) and photographed. were performed and the results were compared with those
of Lelliot & Dicky (1984) and Perombelon & Kelman
Disease control via host fertilization: Different fertilizers (1980). Results showed (Table 1) that majority of the
i.e., Nitrogen (Urea), Phosphorus (Diammonum phosphate) candidate Ecc isolates did not produce (whereas majority
and Potash (Sulfate of potash) and their different levels of candidate Eca, and Ech isolates did produce) acid from
(level s = 247 kg ha-1, level 1 = 252 kg ha-1, level 2 = 257 α-methyle glucoside but their reaction to the production
kg ha-1, level 3 = 262 kg ha-1 each) were evaluated (autumn,
of reducing substances from sucrose was variable.
2008) for their effect in controlling blackleg and soft rot of
However, majority of Eca isolates produced reducing
potato. The experiments were conducted using RCBD
substances from sucrose. These results are in line with
(replicated three times) having 27 treatments and a control.
Potato cultivar Kuroda was used in the trial. Naturally those of Lelliot & Dicky (1984) and Perombelon &
infested field (having the left-over diseased plant debris, Kelman (1980).
discarded rotten tubers and possibly pathogen-harbouring Sensitivity to erythromycin is also a variable
solanaceous weeds as sources of inoculum) in district character. Majority of our strains were not sensitive to this
Mardan was chosen for these experiments. The total antibiotic, some were sensitive and some were weakly
amount of nitrogenous fertilizer used per season was split sensitive (Table 1). El-Hendawy et al., (2002) and Lelliot
into two parts: first half dose of nitrogen fertilizer was & Dicky (1984) found their Eca and Ecc strains not to be
applied before planting and second half dose at the tuber sensitive to erythromycin but Perombelon & Kelman
formation/earthing up stage while the other two fertilizers (1980) reported that their Eca and Ecc strains were
were applied with sowing. Before sowing, seed tubers were sensitive to this antibiotic.
2280 HAFIZ FARHAD ALI ET AL.,
CHARACTERIZATION OF THE CAUSAL ORGANISM OF BLACKLEG ROT OF POTATO 2281

Fig. 1. Growth of Erwinia carotovora subspecies carotovora (A), Erwinia carotovora subspecies atroseptica (B), and Erwinia
chrysanthemi (C) on nutrient agar 48 hours after incubation.

Fig. 2. Potato tissue maceration/soft rot produced by Erwinia carotovora subspecies carotovora (A), Erwinia carotovora subspecies
atroseptica (B), and Erwinia chrysanthemi (C) 24 hours after artificial inoculation on potato halves.

The collected isolates were identified to species or sub- band of 550 bp (Fig. 3B). A few isolates were identified
species level. Most isolates were found to have expected to be Ech because Ech-specific primers amplified the
characteristics. However, some of them showed un- expected 420 bp band from them (Fig. 3C).
expected behavior. For example, all our strains were able to The fact that we can successfully and accurately
grow at 36oC which is the characteristic of Ecc and Ech identify the potato-blackleg causal organisms using specific
group according to Lelliot & Dicky (1984) and Perombelon primers and our optimized PCR conditions has important
& Kelman (1980) and not that of Eca group. Based upon implications for seed certifying agencies doing seed-potato
their growth at 36oC, our Eca strains appear to be atypical, indexing. For this purpose, random samples can be taken
similar to those reported by other researchers (Thomson et from seed-potato lots, crushed in 0.85% saline, plated on
al., 1981; Helias et al., 1998). El-Hendawy et al., (2002) NA medium, DNA extracted (Wang et al., 1993) and PCR-
also reported that 21 of their Eca strains were able to grow tested for the amplification of the 690 bp Eca-specific, 550
at 36oC. Jabuonsky et al., (1986a) reported that bp Ecc-specific, and 420 bp Ech-specific DNA bands. If
unexpectedly 44 of their Ecc strains were not able to grow only one type of disease-causing bacterium is expected to
be present in seed potatoes, single PCR reaction can be
at 37oC. Our strains slightly deviate from the standard
carried out. However, if two or three types of bacteria
description in some of their minor characteristics but they
(causing blackleg and soft rot) are expected; multiplex PCR
still remain the valid members of the Eca group as similar reaction could be performed.
variations in minor characteristics have been found by other
researchers. The occurrence of intermediate forms of Eca Disease management via host fertilization
and Ecc (sharing some of the characteristics of both the
groups) indicates variability happening among these Yield (g.plant-1): Significant differences (p≤0.05) were
strains. This variability poses a threat towards breaking the found in potato yield (g.plant-1) among different
host resistance by the pathogen. combinations of nitrogen (N), phosphorus (P) and potash
(K) fertilizers (Table 2). The maximum yield (579.50
PCR identification of soft-rot Erwinia spp.: To confirm g.plant-1) was obtained when combination N3P1K3 (i.e.
the precise identity of the SRES isolates, molecular 262/252/262 NPK kg ha-1) of fertilizers was used, followed
studies were done using Eca-specific, Ecc-specific and by treatments N3P2K3 (570 g.plant-1) and N3P3K3 (537.25
Ech-specific primers. The Eca-specific 690 bp band was g.plant-1). The later two combinations showed 1.64% and
amplified from a total of 20 isolates confirming that they 7.29% decrease in yield, respectively, over that of the best
were Eca (Fig. 3A). Nineteen isolates were found to be combination. The lowest yield (286.25g.plant-1) was
Ecc as the Ecc-specific primers amplified the expected obtained when combination N3P1K1 was used.
2282 HAFIZ FARHAD ALI ET AL.,

Table 2. Effect of different NPK combinations on the % disease incidence, severity and yield per plant of the
potato crop affected with blackleg and soft rot (autumn 2008).
Parameters
S. # Treatments Disease Increase over Disease Increase over Decrease over
Av. yield per
severity the minimum incidence the minimum the maximum
plant (g)
(%) (%) (%) (%) (%)
1. N1 P1 K1 60.250 a 217.1053 83.000 a 253.1915 327.00 fgh 43.57204
2. N1 P1 K2 49.250 cd 159.2105 73.000 bc 210.6383 337.25 eh 41.80328
3. N1 P1 K3 39.750 f 109.2105 67.000 d 185.1064 350.50 dh 39.51682
4. N1 P2 K1 56.000 b 194.7368 78.000 ab 231.9149 361.25 dh 37.66178
5. N1 P2 K2 46.750 de 146.0526 65.000 de 176.5957 419.25 cg 27.65315
6. N1P2 K3 37.500 fg 97.36842 61.000 ef 159.5745 366.50 dh 36.75582
7. N1 P3 K1 48.000 de 152.6316 74.250 bc 215.9574 330.63 fgh 42.94564
8. N1 P3 K2 30.000 jk 57.89474 61.000 ef 159.5745 431.25 cg 25.5824
9. N1 P3 K3 25.250 mn 32.89474 61.000 ef 159.5745 436.00 cg 24.76273
10. N2 P1 K1 52.000 c 173.6842 61.750 e 162.766 355.75 dh 38.61087
11. N2 P1 K2 47.000 de 147.3684 51.000 ghi 117.0213 431.25 cg 25.5824
12. N2 P1 K3 35.750 gh 88.15789 48.000 ij 104.2553 470.25 ad 18.85246
13. N2 P2 K1 48.000 de 152.6316 64.500 de 174.4681 340.25 eh 41.28559
14. N2 P2 K2 33.000 hi 73.68421 55.000 g 134.0426 417.50 cg 27.95513
15. N2 P2 K3 27.000 lm 42.10526 49.250 hi 109.5745 513.75 abc 11.34599
16. N2 P3 K1 45.500 e 139.4737 60.250 ef 156.383 336.25 eh 41.97584
17. N2 P3 K2 31.000 ij 63.15789 47.000 ij 100 435.25 cg 24.89215
18. N2 P3 K3 28.250 jkl 48.68421 43.250 jk 84.04255 536.75 abc 7.377049
19. N3 P1 K1 46.750 de 146.0526 56.000 fg 138.2979 286.25 h 50.60397
20. N3 P1 K2 28.250 jkl 48.68421 52.000 ghi 121.2766 451.00 bf 22.17429
21. N3 P1 K3 19.000 p 0 23.500 l 0 579.50 a 0
22. N3 P2 K1 37.750 fg 98.68421 53.500 gh 127.6596 312.50 gh 46.0742
23. N3 P2 K2 25.250 mn 32.89474 43.250 jk 84.04255 460.25 ae 20.57808
24. N3 P2 K3 20.500 op 7.894737 39.750 k 69.14894 570.00 ab 1.639344
25. N3 P3 K1 36.500 g 92.10526 49.250 hi 109.5745 428.50 cg 26.05695
26. N3 P3 K2 27.500 klm 44.73684 25.000 l 6.382979 515.25 abc 11.08714
27. N3 P3 K3 23.000 no 21.05263 52.000 ghi 121.2766 537.25 abc 7.290768
NsPsKs 46.750 de 146.0526 72.250 c 207.4468 330.75 fgh 42.92494
LSD(p≤0.05) 2.981 5.0597 124.890
N1, P1, K1 are 252; N2, P2, K2 are 257, and N3, P3, K3 are 262 Kg ha-1, respectively; whereas NsPs and Ks (standard doses of
NPK commonly applied by local farmers) are 247, 247, 247 Kg ha-1 Kg ha-1, respectively. The experiment was repeated once with
similar results

A B C

Fig. 3. Agarose gel (2%) showing expected PCR-bands of 690 bp amplified by Ecc-specific primers (A), 550 bp amplified by Eca-
specific primers (B), and 420 bp amplified by Ech-specific primers (C), PC = Positive control (using DNA template of identified
bacterium), U = Unidentified collected bacterial sample NC = Negative control (no template used), M = Marker. Ecc = Erwinia
carotovora subspecies carotovora, Eca = Erwinia carotovora subspecies atroseptica, and Ech = Erwinia chrysanthemi.
CHARACTERIZATION OF THE CAUSAL ORGANISM OF BLACKLEG ROT OF POTATO 2283

Disease severity and incidence (%): The % disease severity synthesis of secondary compounds such as phenols and
was almost inversely proportional to the yield (Table 2). For quinines. This situation weakens the plant’s natural ability to
example, the combination N3P1K3 (which gave the best yield) defend itself against diseases. Under N limited conditions,
allowed the minimum % diseases severity (19%). This was however, much more C from the Krebs cycle is available for
followed by N3P2K3 (the second best yielder) in which case the synthesis of phenolic compounds. Prokkola (1994) used
the % disease severity was 20.05%, showing an increase of three different levels of nitrogenous fertilizer and found that
7.89 % than that of the minimum. Likewise, N3P3K3 when the disease incidence was high, the proportion of
allowing 23% disease severity, showed 21.05 % increase blackleg stems increased with increasing dozes of N
over the minimum. Maximum % disease severity (60.25%) fertilizer. However, this was not the case when disease
was present in plants receiving the combination N1P1K1, incidence was low. Kumar et al., (1991) also found that
increasing dozes of nitrogenous fertilizers increased storage
showing an increase of 217.10% over that of the minimum.
rots in six potato cultivars they tested. Although, our results
Likewise, in other combinations potash (K) seemed to play a
did not show any significant increase in blackleg severity
role in reducing % disease severity. For example, the % beyond P2 level of phosphorus, Gracia et al., (2004) reported
disease severity in N3P3K2 and N3P3K3 increased by 44.74 that P caused a significant increase in soft rot of tubers. They
and 21.05% over the minimum suggesting that the higher also found that the plant tissue-degrading enzymes
level of potash reduced % disease severity. (polygalacturonase and pectate lyase) of the soft rot-bacteria
However, the % incidence followed somewhat different became more active when bacteria were grown in the
pattern as compared to the % disease severity. Plants in presence of phosphorus.
treatment N3P1K3 (that gave the best yield and allowed As obvious from our studies, potash played an important
minimum % disease severity) showed the lowest diseases role in decreasing blackleg and soft rot severity and
incidence (23.05). It was followed by treatment N3P3K2 increasing yield. Potash might have had some effect on the
which ranked 5th and 6th instead of 2nd in yield and disease pathogen or on the host or both. The effect of potash on the
severity respectively.
host might have been the cross-linking and strengthening of
Means followed by the same letter(s) in the same
the host cells as has been suggested for the effect Ca on host
column are not significantly different from one another at
0.05 level of probability. cell walls (Flego, 1997). In case of the pathogen, potash
Host fertilization plays an important role in disease might have played role in depressing the pathogen genes
severity and is therefore, worth investigating. The types as responsible for the production of bacterial enzymes that
well as the dozes of different chemical fertilizers can either degrade host plant tissue. Potassium is involved in plant
increase or decrease the severity of a disease depending upon enzyme activation, cation/anion balance, stomatal
the disease and host. Absolutely no work has been done in movement, phloem loading and photosynthate translocation
Khyber Pakhtunkhwa on any aspect of potato bacterial and turgor regulation. Photosynthesis is increased with
diseases including the role of fertilizers. We therefore, increasing K content of the leaves. K deficiency can result in
decided to investigate whether or not slight increase (beyond cracks and lesions on the surface of leaves and fruits
the standard dozes) in the dozes of N, P and K would have providing easy access to fungi and bacteria.
any effect on the control of potato blackleg and soft rot. Our results indicated that disease severity increased with
Results of the present study indicated that all the three the increasing levels of nitrogen (Urea: (NH2)2CO) and then
fertilizers (N, P, K) had effect on disease severity but yield it dropped at the N3 level. One possibility that might explain
was affected only by N and K and disease incidence by P our results may be that the variety (kuroda) we used for our
and K. With the increasing levels of N (beyond the standard experiments had some resistance against potato blackleg.
level), there was initially some significant increase in yield This genetic resistance of the variety might have resisted the
but later it became non-significant. Increasing levels of P increase of disease severity with the increasing levels of
fertilizer did not result in any significant increase in the yield. nitrogen. Similar results were reported by Canaday & Wyatt
Increasing levels of potash from K1 to K3 also did not result (1992) who found that side dress applications of ammonium
in any statistically significant increase in potato yield. The nitrate increased the incidence and severity of bacterial soft
effect of increasing levels of nitrogen and phosphorus rot (caused by Pseudomonas marginalis) in a susceptible
fertilization of potato plants on blackleg and soft rot broccoli cultivar, Premium Crop, but had no effect on a
incidence and severity followed almost the same pattern that resistant one, Shogun. McGovern et al., (1985) reported
was observed for yield. However, increasing levels of potash results similar to ours. They found that the susceptibility of
did show significant decrease in both severity and incidence Chrysanthemum morifolium to Erwinia chrysanthemi
of potato blackleg disease. Increasing levels of potash did increased with the increasing rate of fertilizer and nitrogen in
show some significant increase in yield especially at the P2 the form of (NH4)2SO4. However, the susceptibility was
level of phosphorus and significant decrease in blackleg maximum at moderate levels (100-200 ppm) of Ca (NO3)2
severity at all levels of phosphorus. and NH4NO3 and decreased when nitrogen was increased up
Excessive amounts of nitrogenous fertilizers increase to 400 ppm in both forms.
the succulent tissue in potato plants making them more The finding that nitrogen level can be increased up to a
susceptible to diseases especially bacterial diseases (Agrios, point at which disease severity starts decreasing has
2005). The excessive vegetative growth due to overdose of N important implications. Increased nitrogen level will increase
also creates micro-climatic conditions (such as more yield but will not increase disease severity if a proper type of
humidity) favorable for fungal and bacterial diseases. nitrogenous fertilizer is used. Therefore, it is suggested that
Moreover, in case of ample N supply, there is a high demand all the available types of nitrogenous fertilizers be tested to
for carbon (C) from photosynthesis via the Krebs cycle for find out the one that can be used at higher doses to increase
soluble organic compounds, leaving little carbon for the yield without increasing blackleg severity.
 2284 HAFIZ FARHAD ALI ET AL.,

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(Received for publication 15 May 2013)

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