Detection of mycosphaerella using the polymerase chain reaction

Details Detection of mycosphaerella using the polymerase chain reaction Detection of mycosphaerella using the polymerase chain reaction

2001-09-24

2004-05-11

Sitton, Jehanne S. (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06733972

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the use of primers in polymerase chain reaction assays for the detection of a heretofore unknown species of the banana pathogen Mycosphaerella. The use of these primers enables the detection of specific isolates of fungal pathogens and the monitoring of disease development in plant populations.
BACKGROUND OF THE INVENTION
Diseases in plants cause considerable crop loss from year to year resulting both in economic deprivation to farmers and, in many parts of the world, to shortfalls in the nutritional provision for local populations. The widespread use of fungicides has provided considerable security against plant pathogen attack. However, despite $1 billion worth of expenditure on fungicides, worldwide crop losses amounted to approximately 10% of crop value in 1981 (James, 1981
; Seed Sci. & Technol.
9: 679-685).
The severity of the destructive process of disease depends on the aggressiveness of the pathogen and the response of the host. One aim of most plant breeding programs is to increase the resistance of host plants to disease. Typically, different races of pathogens interact with different varieties of the same crop species differentially, and many sources of host resistance only protect against specific pathogen races. Furthermore, some pathogen races show early signs of disease symptoms, but cause little damage to the crop. Jones and Clifford (1983; Cereal Diseases, John Wiley) report that virulent forms of the pathogen are expected to emerge in the pathogen population in response to the introduction of resistance into host cultivars and that it is therefore necessary to monitor pathogen populations. In addition, there are several documented cases of the evolution of fungal strains that are resistant to particular fungicides. As early as 1981, Fletcher and Wolfe (1981
; Proc.
1981
Brit. Crop Prot. Conf.) contended that
24% of the powdery mildew populations from spring barley and 53% from winter barley showed considerable variation in response to the fungicide triadimenol and that the distribution of these populations varied between varieties, with the most susceptible variety also giving the highest incidence of less susceptible types. Similar variation in the sensitivity of fungi to fungicides has been documented for wheat mildew (also to triadimenol), Botrytis (to benomyl), Pyrenophora (to organomercury), Pseudocercosporella (to MBC-type fungicides) and
Mycosphaerella fijiensis
to triazoles to mention just a few (Jones and Clifford; Cereal Diseases, John Wiley, 1983).
There are two well-known forms of sigatoka leaf spots which affect bananas—yellow sigatoka, caused by
Mycosphaerella musicola
and black sigatoka caused by
Mycosphaerella fijiensis
. Black sigatoka is the more economically devastating, causing causes significant reductions in leaf area, yield losses of 50% or more, and premature ripening, a serious defect in exported fruit . It is more damaging and difficult to control than the related yellow sigatoka disease, and has a wider host range that includes the plantains and dessert and ABB cooking bananas that are usually not affected by yellow sigatoka.
In export plantations, black sigatoka is controlled with frequent applications of fungicides. This is a very expensive practice because it includes the use of airplanes or helicopters, permanent landing strips and facilities for mixing and loading the fungicides, and the high recurring expense of the spray materials themselves. In total, it has been estimated that these costs are ultimately responsible for 25% of the final retail price of these fruit in the importing countries (www.scisoc.org/feature/banana/top.html as found on Apr. 17, 2000). Different sterol demethylation inhibitors (DMIs) are now the most commonly used compounds for the control of sigatoka, but increased tolerance of the pathogen to the DMI fungicides has made it necessary to increase applications in several countries in banana-growing regions to frequencies of 25-40 per year (www.scisoc.org/feature/banana/top.html as found on Apr. 17, 2000).
Although black sigatoka can often be recognized visually, unambiguous diagnosis can be complicated by the presence of other pathogens found on banana leaves. Isolation of the pathogen, which is most successfully achieved by ascospore discharge from necrotic leaf material, is often confounded by the absence of mature perithecia, and even when obtained in culture, I M. fijiensis and
M. musicola
are not readily visually differentiated (Johanson and Jeger
Mycol. Res.
97 (6): 670-674 (1993)).
Biomedical researchers have used PCR-based techniques for some time and with moderate success to detect pathogens in infected animal tissues. More recently, however, this technique has been applied to detect plant pathogens. The presence of
Gaumannomyces graminis
in infected wheat has been detected using PCR of sequences specific to the pathogen mitochondrial genome (Schlesser et al., 1991
; Applied and Environ. Microbiol.
57: 553-556), and random amplified polymorphic DNA (i.e. RAPD) markers were able to distinguish numerous races of
Gremmeniella abietina
, the causal agent of scleroderris canker in conifers. U.S. Pat. No. 5,585,238 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of
Septoria tritici, Septoria nodorum, Pseudocercosporella herpotrichoides
(R- and W-types),
Mycosphaerella fijiensis
, and
Mycosphaerella musicola
and their use in the identification of these fungal isolates using PCR-based techniques. In addition, U.S. Pat. No. 5,955,274 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Fusarium and their use in the identification of these fungal isolates using PCR-based techniques. Furthermore, U.S. Pat. No. 5,800,997 (herein incorporated by reference in its entirety) describes primers derived from the ITS sequences of the ribosomal RNA gene region of strains of Cercospora, Helminthosporium, Kabatiella, and Puccinia and their use in the identification of these fungal isolates using PCR-based techniques.
In view of the above, there is a real need for the development of technology that will allow the identification of additional specific races of pathogen fungi early in the infection process. By identifying the specific race of a pathogen before disease symptoms become evident in the crop stand, the agriculturist can assess the likely effects of further development of the pathogen in the crop variety in which it has been identified and can choose an appropriate fungicide if such application is deemed necessary.
SUMMARY OF THE INVENTION
The present invention pertains to methods of identification of different pathotypes of plant pathogenic fungi. The invention provides Internal Transcribed Spacer (ITS) DNA sequences that show variability between different fungal pathotypes. Such DNA sequences are useful in the method of the invention as they are used to derive primers for use in polymerase chain reaction (PCR)-based diagnostic assays. These primers generate unique fragments in PCR reactions in which the DNA template is provided by specific fungal pathotypes and is thus used to identify the presence or absence of specific pathotypes in host plant material before the onset of disease symptoms.
In particular, the present invention provides the Internal Transcribed Spacer (ITS) DNA sequences from a heretofore unknown species of Mycosphaerella, as well as ITS-derived diagnostic primers for the detection of this species of Mycosphaerella and for differentiating it from other Mycosphaerella species such as
Mycosphaerella fijiensis
and
Mycosphaerella musicola.
In one embodiment, the present invention provides a DNA molecule isolated from the ribosomal RNA gene region of a fungal pathogen, wherein said DNA molecule is the Internal Transcribed Spacer (ITS) DNA sequence of a heretofore unknown species of Mycosphaerella. In a preferred embodiment, the

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