1. IntroductionStubborn disease of citrus is one of the most important graft-transmissible diseases affecting citrus trees, particularly in warm climatic regions. It is caused by the prokaryote Spiroplasma citri, which is a plant pathogenic bacterium belonging to the class Mollicutes, a group of microorganisms phylogenetically related to low G-C content Gram-positive bacteria [1]. Spiroplasma citri was first reported in Egypt by Nour El-din in 1967 [2], who described the symptoms of the disease and suggested that the causal agent was graft-transmissible. Later, it was isolated and cultured [3].
Citrus is considered to be the main fruit crop in Egypt. The annual production of orange fruit, as the most important citrus variety, averages 3.7 million tons in 2011 [4], equaling 37.7% of the total fruit trees production. Citrus is produced mainly in the Nile Delta and valley (old lands), where nearly 71% of the total citrus area in this region is located [5]. In 2011, although production was forecast up in Egypt, exports were forecast downward due to uneven quality [4]. This drew attention to the epidemiology and the precise detection of the quality diseases in the area, such as citrus stubborn disease (CSD), which would help with the management of such disease.
It was noticed on several previous reports that the rate of greenhouse transmission of Spiroplasma citri, determined by the traditional method of biological indexing on the indicator plant, is relatively low and that the time required for the symptoms’ appearance on the indicator plant is long. This could be due to the low concentration of the pathogen in the budstick used specially during the cold seasons; therefore, the traditional method of biological indexing needs to be modified in such a way as to overcome the problems of the low rate of successful green-house transmission.
2. Material and Method2.1. Field Observation and SamplingFirst, Observations in various fields were carried out at different times of the year (October 2010, May, July and August 2011), in order to check for symptoms of CSD. During the survey, only symptomatic samples were considered. Further, three symptomatic and off-season fruits were collected per suspected tree. A total of 130 samples were collected at different times of the year: 100 samples belonging to three orchards at Al Qalyoubia (Washington navel orange and sweet orange), and 30 samples belonging to the experimental orchard at the Faculty of Agriculture, Cairo University (Sweet orange).
The confirmation of the visual symptoms observed was carried out by culturing in LD8 media and microscopic observation of the S. citri.
2.2. Isolation and CulturingPetiole leaf midribs or fruit columella were excised, surface disinfested and diced with a sterile razor blade in 5 ml of LD8 broth medium [6], passed through a 0.45-µm filter, and incubated at 30˚C. The presence of S. citri was confirmed after 3 to 14 days by examining 10 µl of culture medium by dark-field microscopy at ×400 to 1000 for the presence of motile, helical spiroplasma.
2.3. Biological IndexingThree different methods of biological indexing (traditional method, inoculated indicator and inverse inoculation) were conducted and compared in order to evaluate the most efficient method and the most suitable conditions for transmission of the S. citri in the greenhouse. For that, young shoots with small emerging leaves from symptomatic trees were collected to be used in these trials.
Positive control trees, as well as negative control from healthy trees, were also included in the tests. Different aspects were evaluated, including the successful transmission rate and the time required for the appearance of stubborn symptoms on the indicator plant.
2.3.1. Traditional MethodAs reported by Roistacher in 1991 [7], side grafting and leaf batch grafting were performed; collected budwood (stem pieces that were 5 - 7 mm in diameter from Stubborn infected trees) were side-grafted onto the one-yearold Madame Vinous seedlings (stubborn indicator plant). After sealing the graft with parafilm, and then labeling and enclosing it inside a plastic bag, the inoculated plants and the negative controls were maintained in a warm, conditioned greenhouse. After 1 - 2 weeks, the plastic bags were opened at the top to reduce humidity and observations of the symptoms were carried out weekly.
2.3.2. Inoculated IndicatorWood cuttings containing 4 - 6 nodes of the indicator “Madame Vinous” were firstly inoculated by chip budding or by leaf inoculation using the bark tissue collected from the sample sources; then, the inoculated indicator cuttings were grafted onto Sour orange rootstock (Figure 1) and enclosed inside a plastic bag in order to keep high humidity inside. The inoculated plants were maintained in a warm conditioned greenhouse. After ten days, the plastic bags were opened at the top in order to reduce the inside humidity, grafting success was evaluated and symptoms observations were carried out weekly.
2.3.3. Inverse InoculationThis new technique was set up in order to overcome the limitations of the traditional indexing related to the low concentration of S. citri in the stubborn infected sample and the long time needed for the diffusion of the pathogen in the plants. This method is the reverse of the inoculated indicator method, as it consists of the inoculation of a cutting from the sample to be tested with a bud from Madame Vinous sweet orange indicator, and then the obtained inoculated cutting was grafted onto sour orange rootstock (Figure 2). Symptoms observations were carried out weekly on leaves emerging from the indicator bud.
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Figure 1. The Inoculated Indicator method.
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Figure 2. The Inverse Inoculation method.