PCR primers for detection and identification of plant...

Details PCR primers for detection and identification of plant... PCR primers for detection and identification of plant...

2000-11-02

2002-07-23

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S004000, C435S005000, C435S091100, C435S091200, C435S069100

Reexamination Certificate

active

06423499

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The species
Acidovorax avenae
, formerly classified as
Pseudomonas avenae
, contains different subspecies pathogenic to several hosts including oat, corn, millet, wheat, sugarcane, rice, orchid, konjaci, and Cucurbitaceae plants including melon, watermelon, and squash. This invention relates to novel PCR primers which can be used to detect plant pathogenic
Acidovorax avenae
species and to distinguish among the pathogenic subspecies and strains, particularly to distinguish (1)
A. avenae
from other bacteria, (2)
A. avenae
subsp.
citrulli
(infects only Cucurbitaceae) from other subspecies and (3) rice strains of
A. avenae
subsp.
avenae
from other strains originating from other plants as well as from other subspecies of
A. avenae.
2. Description of the Relevant Art
The bacterium
A. avenae
subsp. avenaecauses bacterial stripe of rice, leaf blight of oats, red stripe disease of sugarcane and millet, and brown stripe of
Setaria italica
. The organism was originally described as
Pseudomonas avenae
by Manns in 1909 (
Ohio Agric. Exp. Stn. Res. Bull
. 210: 91-167) as the causal agent of leaf blight on oats in Ohio. Another organism,
P. alboprecipitans
, described by Rosen in 1922 on foxtail (
Ann. Mo. Bot. Gard
. 9:333-402) and on corn in 1926 (
Phytopathology
16:241-267) was later shown to be synonymous with
P. avenae
(Schaad et al. 1975
. Int J. Syst Bacteriol
. 25:133-137). On rice, the organism has been previously referred to as
P. setariae
or
P. panici
(Goto. 1964
. Bull. of Faculty of Agriculture
, Shizuoka University 14: 3-10) and on sugarcane as
P. rubrilineans
(Martin et al. 1989.), the causal agent of red stripe diseases of sugarcane (C. Ricaud et al. (Eds), Elsevier, The Netherlands, pp. 80-95).
Acidovorax avenae
subsp.
citrulli
causes bacterial blight and fruit blotch of watermelon and melon. The organism was originally described by Schaad et al. (1978
. Int. J. Syst. Bacteriol
. 28:117-125) in plant introduction breeding lines of watermelon in Georgia as
P. pseudoalcaligenes
subsp.
citrulli
. A closely related organism,
A. avenae
subsp.
cattleyae
occurs on orchids (Ark and Thomas. 1946
. Phytopathology
36:695-698) and
A. konjaci
(Goto, M. 1983
. Int J. Syst. Bacteriol
. 33:539-545) occurs on konjac (
Amorphophalus konjaci
Koch).
As the host range of
A. avenae
subsp.
avenae
is wide to monocotyledonous and dicotyledonous plants, it has been thought to be polyxenic. However, individual strains of the pathogen infect only one or a few host species (Kadota, I. 1996
. Bulletin of the Hokuriku National Agricultural Experiment Station
38:113-171; Nishiyama et al. 1979
. Ann. Phytopathol. Soc. Jpn
. 45:25-31). For example, rice strains can infect rice but not other monocotyledonous plants including finger millet, whereas finger millet strain cannot infect rice plants. Furthermore, the heterogeneity of rice strains and other plant strains of the bacterium has been suggested based on the biochemical characteristics and the protein profiles of the cells. Even though the reclassification of previous
P. avenae
strains to
A. avenae
subsp.
avenae
has been widely accepted, a certain degree of pathogenic, serological, and genomic, as well as phenotypic, heterogeneity still exists among reported strains of
A. avenae
subsp.
avenae
subspecies. Pathogenicity of
A. avenae
subsp.
avenae
strains specific for rice particularly differs from non-rice strains of
A. avenae
subsp.
avenae
. It has been thought, therefore, that rice strains of
A. avenae
subsp.
avenae
are different from other plant strains. However, hitherto there is no sensitive and specific means which can detect all the strains of
A. avenae
subsp.
avenae
which have originated from different hosts and thus satisfy the current taxonomic situation at the subspecies level.
Phylogenetically,
A. avenae
is included in the new “acidovorans” DNA-rRNA homology group (Willems et al. 1992
. Int J. Syst Bacteriol
. 42:107-119). This group contains many pathogens previously classified in the non-fluorescent pseudomonad group, including
P. avenae
Manns 1909 (
Ohio Agric. Exp. Stn. Res. Bull
. 210:91-167),
P. cattleyae
Savulescu 1947 (Pavarino. 1911
. Atti Accad. Lincei
20:233-237),
P. pseudoalcaligenes
subsp.
citrulli
(Schaad et al. 1978, supra),
P. pseudoalcaligenes
subsp.
konjaci
(Goto, 1983, supra) and
P. rubrilineans
(Lee et al. 1925. Red Stripe Disease. Pamphlet of Hawaiian Sugar Planters Association). Recently, all of these bacteria have been reclassified as subspecies of
A. avenae
, i.e.,
A. avenae
subsp.
avenae, A. avenae
subsp.
cattleyae
, and
A. avenae
subsp.
citrulli
, on the basis of the results of DNA-DNA hybridization, DNA-rRNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests (Willems et al., supra).
A. avenae
subsp.
avenae
is one of the major seedborne bacterial pathogens of rice. Seeds infested with the pathogen are important sources of primary inoculum and a means of dissemination to new areas. Therefore, sensitive and simple detection methods are needed to screen seed lots. Presently, the only reliable method to identify the
A. avenae
pathogens is to isolate them on agar media and confirm their presumptive identification by time consuming and expensive pathogenicity tests. Although a few methods have been used for the detection of
A. avenae
subsp.
avenae
, none has been optimized for selectively detecting the bacterium from rice seeds. Serological methods and a rapid pathogenicity test have been used for routine detection of this pathogen in rice seeds (Kadota et al., supra; Shakya. 1987
. Korean J. Plant Path
. 3:300; Zeigler et al. 1989
. Intl. Rice Res. Newsletter
14:27-28). However, in addition to the issues of specificity and sensitivity, serological techniques have the distinct disadvantage of not being able to provide information on viability and pathogenicity of the seedborne inoculum. In rice,
A. avenae
subsp.
avenae
can be recovered from both diseased, or asymptomatic, apparently healthy seeds. However, identification is difficult, because the organism is frequently overgrown by other bacterial pathogens of rice, such as Pantoea (Erwinia) herbicola, Burkholdera (Pseudomonas) glumae,
B. fuscovaginae
, or
P. syringae
pv.
syringae
. These pathogens are recognized as producing distinct disease symptoms, but field diagnosis is very difficult. In such cases, detection and identification is based on pathogen isolation, through the use of semiselective media, and on pathogenicity tests which discriminate between the various pathogens. However, the presence of coexisting epiphytes such as
P. fulva, P. corrugata, P. putida
, and
P. fluorescens
in/on rice seed makes isolation difficult. Furthermore, rice seedlings are difficult to grow and symptoms on seedlings are often non-discriminating. Several methods of identification of
A. avenae
subsp.
avenae
have been proposed including growth and isolation on semiselective agar media and serology (Shakya, supra). None have achieved much success. The disease cycle, therefore, has not been clarified, because the symptoms are masked after the four or five leaf stage and no adequate techniques are available for monitoring the pathogen in rice plants. Further, there is currently no reliable, routine method available for detecting
A. avenae
in rice seeds. More sensitive and specific methods are needed to confirm the identification, especially in seed health evaluations.
Similar problems exist for watermelon and melons. Although originally described as a seedling disease, watermelon fruit blotch has emerged as a serious disease of mature fruit. Complete losses of production fields often occur due to fruit rot. Like most seedborne bacteria, control is based primarily on seed health testing. Because of the seriousness of watermelon fruit blotch, nearly all watermelon seed lots must be assayed for
A. avenae
subspec.
citrulli
. This involves

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