Download Assessment of the sanitary status of pome fruit crops in Kosovo, with

ORIGINAL SCIENTIFIC PAPER
Assessment of the sanitary status of pome
fruit crops in Kosovo, with particular
emphasis to the bacterial disease the Fire
Blight
Naim KRASNIQI1, Arben MUSLIU1, Kujtim LEPAJA2, Lavdim LEPAJA2
University of Prishtina, Faculty of Agriculture and Veterinary Science, Boulevard Bill Clinton N.N., 10000 Prishtina,
Kosovo, (e-mail: [email protected])
2
Agricultural University of Tirana, Horticultural Department, Koder-Kamëz, 1029 Tirana, Albania
1
Abstract
Pome fruits represent a very important fruit crops in Kosovo, covering around 50% of the
total fruit production. Economic losses induced by Erwinia amylovora were reported in
neighbouring countries. In order to understand the real situation of the bacterial diseases
on pome fruits in the Kosovo, assessment was carried out for detecting 3 bacteria (E.
amylovora, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. papulans) on
apple and pear. Morphological, biochemical (LOPAT test) and molecular (rep-PCR) tests
were performed. This survey showed that several bacteria were present and in some cases
widely distributed. Furthermore, this current sanitary status could be worsened due to the
wide distribution of E. amylovora, which seems to be widely distributed on apple and pear
in the different cultivated areas of the country.
Key words: morphological and biochemical tests, Erwinia amylovora, Kosovo, pome fruits,
rep-PCR
Introduction
In Kosovo there are good conditions of remarkable topography, enriched soil, enough water available,
sufficient sun exposure, adequate altitude and fresh mountainous breeze that have acceded the Kosovo
pome fruits their outstanding features. In fact apple in Kosovo ranks the first in the total fruit production,
while pear is grown to a lesser extent and quince groves are very limited to non-commercial level. In
order to maximize their outputs with the least expenses possible, growers were constrained to use their
own propagating material. This was accompanied by a lack of knowledge of the existence of diseases and
topped by a shortage of an efficient certification program. Therefore, infected material was distributed and
introduced into the country and freely circulated. Today the world is confronted with plant diseases, most
of which are asymptomatic and spread in a latent way. To assess the presence and spread in the field of
most of the agents of these diseases, specific detection techniques were brought to light.
Material and methods
Field surveys and collection of the samples. The first survey and samples collected was carried out during
winter 2012. Observations on symptoms associated to bacterial infection on apple and pear trees were
traced. It took place in Prishtina and Kamenica areas. The second survey was carried out by the end of June
2012, in Peja, Mitrovica and Ferizaj localities (Figure 1). Almost all samples collected in Peja and Ferizaj
areas, were showing symptoms which can be associated to the bacterium Erwinia amylovora infection,
while in Ferizaj asymptomatic samples were collected. A total of 100 samples from apple and pear trees
were collected among mother plots and commercial orchards during the surveys.
Proceedings . 50th Croatian and 10th International Symposium on Agriculture . Opatija . Croatia (605–612)
Section 9 . Pomology
605
Naim KRASNIQI, Arben MUSLIU, Kujtim LEPAJA, Lavdim LEPAJA
Figure 1. Map of inspected areas related to
bacteria surveys
Characterisation to the genus level. All isolates were first characterized to the genus level. To confirm the
identity of the isolates to the genus Erwinia and Pseudomonas spp., some key tests were performed, in case of
asymptomatic and symptomatic samples (Figure 2).
a
b
Figure 2. The scheme for the detection and identification of Erwinia amylovora in asymptomatic (a) and
symptomatic samples selected (b) (EPPO Protocol, 2004).
Morphological tests on the cell morphology were carried out by using a. KOH test 3.0% (w/v). This technique
is simple and rapid and can be used for identification and separation of bacteria into two broad groups: Grampositive and Gram-negative. It is considered as a useful method and can be used as a supplementary test to
Gram staining in cases where the latter gives rise to doubt (Gregersen, 1978). While colonial morphology was
carried out by using NAS, KB and CCT media; the isolates were streaked separately onto Petri plates containing
King’s B, (King et al., 1954), Nutrient agar sucrose (NAS) and CCT media. The bacteria were streaked onto
those media and incubated at 27oC. After 48 h, observations of the morphological properties were done for the
production of fluorescent pigment into King B medium (King et al., 1954), the levan production in Nutrient
Agar Sucrose (NAS) medium, and formation of typical colonies, mucoid with shiny surface, semi-transparent
and slight violet into CCT medium (Schaad et al., 2001).
Molecular tests were performed by rep - PCR (repetitive-sequence based Polymerase Chain Reaction).
Repetitive sequence-based PCR or rep-PCR is based on amplification from the sites of repetitive extragenic
paliandromic (REP) elements, Enterobacterial repetitive intergenic consensus (ERIC) sequences and BOX
elements found at different positions on the bacterial genome. Subsequently, three families of repetitive
sequences could be used including 35-40 bp repetitive extragenic paliandromic (REP) sequence, the 124-127
bp enterobacterial repetitive intergenic consensus (ERIC) sequence, and the 154 bp BOX element (Versalovic
et al., 1991). These sequences are located in distinct, intergenic positions all around the chromosome. REP and
ERIC have been described only for Gram-negative enteric bacteria and closely related phyla (Versalovic et al.,
606
50th Croatian and 10th International Symposium on Agriculture
Assessment of the sanitary status of pome fruit crops in Kosovo, with particular emphasis to the bacterial
disease the Fire Blight
1991; 1994), whereas the BOX primer has been described only in the Gram-positive Streptococcus pneumonia
(Martin et al., 1992). This technique is simpler and faster for the analysis of large numbers of strains, although
it is less discriminative. This type could be used to design highly specific molecular markers for strain typing
which could have epidemiological significance to trace the origin and routes of dispersal of a given outbreak
(Rico et al., 2007). DNA extraction bacterial cultures (1.5 mL) grown overnight in Luria Bertani broth
(LB) at 28°C were centrifuged at 13.000 rpm for 2 min and the supernatant was discarded. Whole genome
DNA extraction was performed by using the GenElute Bacterial Genomic DNA Kit (Sigma), following the
manufacturer’s recommendations.
Results and discussion
Collection of E. amylovora and Pseudomonas spp. isolates. The surveys were carried out in two periods; in
January and late June 2012. Over 40 Erwinia amylovora and Pseudomonas spp. suspected samples were collected
from apple and pear mother plots and commercial orchards in Prishtina, and Kamenica during January 2012,
while the second survey was carried out in late June – early July respectively; 60 isolates from Peja, Mitrovica
and Ferizaj. In Peja and Ferizaj areas, almost all samples collected, were symptomatic to the bacterium Erwinia
amylovora, while in Mitrovica symptomless samples were collected, and brought at Mediterranean Agronomic
Institute of Bari (MAIB) for further laboratory assays.
b
a
Figure 3. (a and b) Field
observations during the
surveys
Typical symptoms of Fire Blight disease were observed such: leaf blight and hook shape formation likely to
be Erwinia amylovora; leaf blight on apple shoot (Figure 3a); the blight on pear leaf, tip of the shoot typically
curved downward in a hook shape (Figure 3b).
Section 9 . Pomology
607
Naim KRASNIQI, Arben MUSLIU, Kujtim LEPAJA, Lavdim LEPAJA
I. Biochemical tests
The biochemical tests were performed to detect E. amylovora and some Pseudomonas spp. that affects pome
fruits. It is important to mention that 21 samples showed the same profile by biochemical tests likely to be E.
amylovora, and the same samples were positive to Erwinia amylovora by rep-PCR as well. The biochemical
tests are showed in details in Table 1.
608
50th Croatian and 10th International Symposium on Agriculture
Assessment of the sanitary status of pome fruit crops in Kosovo, with particular emphasis to the bacterial
disease the Fire Blight
Table 1. The biochemical tests for detection of E. amylovora and Pseudomonas spp.
*a, b – show the presence of more than one bacteria in 1 isolate
Molecular tests
The rep-PCR assay was carried out using REP, BOX and ERIC primers on 70 Kosovan isolates, meanwhile
different strains for Erwinia amylovora and Pseudomonas spp. belonging to different countries and hosts
were used. The amplified genomic DNA fragments obtained on the agarose gel showed the patterns that E.
amylovora isolates obtained from different localities in Kosovo and reference strains from Italy and Algeria
seems to be similar among them and different out-group bacteria.
Section 9 . Pomology
609
Naim KRASNIQI, Arben MUSLIU, Kujtim LEPAJA, Lavdim LEPAJA
Figure 4. rep-PCR fingerprint patterns of genomic DNA from isolates obtained by using REP primer
(M) Molecular marker. Lanes 1 to 21 Kosovar Erwinia amylovora isolates; 22 to 28 suspected Kosovar
Pseudomonas spp. isolates. Lane 29 Italian E. amylovora isolate; lane 30 Algerian E. amylovora isolates; lane
31 P. syringae pv. syringae in Malus domestica (CFBP-5472); lane 32 P. syringae pv. syringae in Pyrus communis
(CFBP-311); lane 33 P. syringae pv. papulans in Malus sylvestris (CFBP-1754) and lane 34 Negative control.
610
50th Croatian and 10th International Symposium on Agriculture
Assessment of the sanitary status of pome fruit crops in Kosovo, with particular emphasis to the bacterial
disease the Fire Blight
Conclusions
Erwinia amylovora was extensively distributed on apple and pear in several localities of Kosovo, confirming
the wide distribution of this bacterium in the Balkan area. On the other hand, Pseudomonas syringae pv.
syringae which was detected molecularly in a single apple tree, constitute the first report of this bacterium
in the country. Older cultivars (Idared, Golden Delicious and Red Delicious) showed to be the most infected
cultivars comparing to the newly introduced cultivars in Kosovo. This work will constitute a basic study on
the prevalence of pome fruit bacterial diseases in Kosovo and will allow the national authorities to establish a
national program to improve the quality of propagative material and the fruit production. The governmental
authorities should: a) take into consideration this first survey and not to allow the circulation of the propagative
material from these infected areas, b) apply strict quarantine measures in order to block the spread of infection
from the neighboring countries, c) constrain the nurserymen to use tolerant cultivars to the Fire Blight.
Acknowledgments
I would like to express my humble gratitude to CIHEAM/IAM-Bari, for the financial assistance during my
Master studies, for providing me an inspiring and stimulating environment for my research. Many thanks
to the all IPM Department staff, for their kind supervision, their enormous help, guidance, patience and
experience they extended to me during this study.
Section 9 . Pomology
611
Naim KRASNIQI, Arben MUSLIU, Kujtim LEPAJA, Lavdim LEPAJA
References
Gregersen T (1978). Rapid method for distinction of Gram–negative from Gram–positive
bacteria. Eur. J. Appl. Microbiol. Biotechnol, n. 5:123–127.
King E.O., Ward M.K., Roney D.E. (1954). Two simple media for demonstration of pyocyanin
and fluorescein. J. Lab. Clin. Med., 44: 301-307.
Martin B., Humbert O., Camara M., Guenzi E., Walker J., Mitchell T., Andrew P., Prudhomme
M., Alloing G., Hakenbeck R., Morrison D.A., Boulnois G.J., Claverys J.P. (1992). A highly
conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.
Nucleic Acids Research, 20 (13): 3479–3483.
OEPP/EPPO (2004). EPPO Bulletin 34, 155 –157.
Rico A., Fuhrer M.E., Ortiz-Barredo A., Murillo J. (2007). Polymerase chain reaction
fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design
of highly specific molecular markers. Phytopathology, 98 (3): 260-269.
Schaad N. W, Jones J. B., Chun W. (2001). Laboratory guide for identification of plant
pathogenic bacteria. Third Edition. Laboratory guide for identification of plant pathogenic
bacteria. APS Press, 1-373.
Versalovic J, Koeuth T., Lupski JR (1991) Nucl. Acids Res. n. 19: 6823-6831
Versalovic J., Schneider M., de Bruijn F.J, Lupski J.R. (1994). Genomic fingerprinting of
bacteria using repetitive sequence based PCR (rep-PCR) Methods in Molecular and Cellular
Biology, 5: 25-40.
sa2015_p0911
612
50th Croatian and 10th International Symposium on Agriculture