USDA-ARS National Clonal Germplasm Repository for Citrus & Dates

‘Stubborn disease of citrus’ was first observed about 1915 in ‘Washington’ navel trees nearRedlands. The first report of stubborn from outside ofCaliforniawas fromPalestinein 1928. Stubborn is now known to be established in most warm, dry inland producing areas inCaliforniaandArizona, and is also a serious disease in most citrus-producing countries with suitable climates. These include countries with arid or semi-arid subtropical climates, but stubborn disease has not been reported from farther east thanIran. In addition, it has not been reported from countries or states with semi-tropical or tropical climates.

The classical concept of citrus stubborn disease involves symptoms expressed in the fruit and in the vegetative growth of citrus trees. Both vegetative and fruit symptoms are often variable and irregularly distributed in the tree.

Fruit symptoms are the most characteristic and the most useful for visual diagnosis in the field. Stubborn-affected trees often flower irregularly (usually in December) and so will have fruits of varying maturity and size present on the tree, although many of the fruit produced on stubborn-affected trees drop while very small. Fruit on stubborn-affected trees produce small fruit that are lop-sided or “acorn-shaped”. The skin of the stylar end of the fruit is thin and subject to early breakdown and split, or to stylar-end greening. Color development is often irregular, and frequently stubborn-affected fruit remain green or yellowish-green. Internally, the flesh is dry and the flavor bitter. In normally seedy varieties, there is extensive seed abortion and/or small, under-developed seeds. Another symptom seen less regularly, and generally only when cutting into the fruit, is a blue albedo.

Trees affected by stubborn have ”bunchy” growth, with shortened internodes and usually upright leaves. Leaves are often smaller and pointier than normal citrus leaves, and sometimes show a mottle resembling zinc deficiency. Stubborn-affected trees lack vigor and do not flush normally. When flushing does occur, it is often greater in the fall than in the spring. These patterns of vegetative growth result in the characteristic flattened top associated with stubborn-affected trees. In addition, there is often leaf drop and sometimes die-back associated with stubborn.

Trees infected with stubborn early in their life, particularly during the nursery phase, are often extremely stunted and may never attain more than 6 feet in height. This is the classical picture of stubborn disease, and veteran researchers have told us that they did not worry much about stubborn since they selected budwood from large, asymptomatic trees and once the trees were established, an infection with the stubborn pathogen did not have much effect. However, during our investigations the last several years, we have observed large, mature trees with sectors that show stubborn symptoms. These sectoral infections result in a decrease in the amount of salable fruit and thus of economic return. We have also observed trees that are smaller than normal having lower than normal production, but not showing the extreme stunting and negligible yield classically associated with stubborn.

Navel oranges and grapefruits are often severely affected.Valenciasgenerally (but not always) show less symptom development than navels but often drop fruit excessively when ripe. Mandarins seem to show vegetative symptoms more readily than they do fruit symptoms, possibly due to the more variable fruit in mandarins in general. Symptoms are harder to detect in lemons and limes.

Although symptoms and effects of stubborn were well established for many years, the causal agent was unknown and was thought to be a virus. However, in the late 1960’s to early 1970’s, the causal organism, Spiroplasma citri, was identified and characterized as a helical, wall-less bacterium motile in liquid and solid cultures. It has an optimum temperature for growth in culture of about 90 °F. S. citri has also been shown to cause various other diseases in crop plants and also affects a number of ornamental plants and several native or invasive species that have become established in California. S. citri is a simple organism with a reduced genome. Most published genes do not reveal any genetic diversity. This is true of the spiralin gene, which is used as the basis of detection by PCR. However, we have detected genetic diversity between stubborn isolates using AFLP.

Diagnosis based upon visual evaluation of symptom expression, particularly fruit symptoms, in severely stubborn-affected trees can be quite reliable. Visual diagnosis of field trees is most effectively done when temperatures are warm and particularly when fruit development is advanced enough that symptoms can clearly be seen (September - October).

In less severe cases of stubborn, positive diagnosis requires confirmation by controlled testing. Biological indexing for stubborn involves graft inoculation of tissue into sensitive varieties, such as ‘Madam Vinous’ or ‘Pineapple’ sweet orange, held at warm temperatures. The most definitive detection technique involves culturing the bacterium from vegetative or reproductive tissue, which requires a number of time-consuming and intricate steps. Growth of S. citri in culture usually takes 2 – 3 weeks and contamination can result in false positives.

Detection by serological techniques has not proven effective in California. Our recent work has resulted in easier and more reliable detection of S. citri using PCR. Initially, we were able to achieve more reliable results from field trees by first putting the appropriate plant parts into culture and then performing PCR on DNA extracted from the medium. We were later able to achieve satisfactory results by performing PCR directly from the culture medium. More recently, we have been able to detect S. citri directly from fruit or vegetative tissue by PCR. At this point, the PCR test is more sensitive than the traditional culture method. This is probably due to the fact that during seasons of low titer, growth of the organism in culture is very slow, whereas amplification of the DNA by PCR is less affected by the low titer.

No matter what actual assay is used, detection of S. citri is made more difficult by its irregular spatial and temporal distribution. All of the testing methodologies listed above are dependent upon the actual presence of the pathogen in the tissue sampled. In the case of S. citri, it cannot be assumed that the pathogen is present in symptomatic trees, nor in all parts of infected trees. Early work demonstrated that stubborn was most detectable during summer months in Riverside. In the San Joaquin Valley, the pathogen becomes routinely detectable slightly later in the year. This is most probably due to winter-time titers of S. citri being lower in trees in the San Joaquin Valley as compared to trees in Southern California due to the colder temperatures in the San Joaquin Valley. When we inoculated greenhouse-grown sweet orange indicators with tissue taken from the San Joaquin Valley in December, it took approximately 6 months until the titer had increased enough under these optimal conditions to be detected. S. citri is also irregularly distributed in infected trees and does not spread systemically with much efficiency. Therefore, if a tree is infected when it has already obtained some size, only the branch or area near the infection will become symptomatic. In randomly sampling symptomatic trees, we have found only about 15 – 20 % of the samples taken test positive. Because of these factors, a sampling strategy for stubborn is critical. Although not conclusive at this point, we recommend that trees should be sampled in the late summer through early fall (July through October). A fairly large sample of approximately 15 budsticks should be sampled, with samples being taken from symptomatic areas if possible.

Stubborn is a graft-transmissible disease, meaning that it can be spread via budwood. However, the graft-transmissibility of S. citri is low and often variable due to the irregular distribution of the pathogen in the tree and the low titers of the pathogen in infected budwood. Because of these factors, a significant proportion of the grafted progeny of an infected tree may be free of S. citri. S. citri has not been shown to be mechanically transmissible nor transmitted by seed. In the 1970s the natural spread of stubborn by insect vectors was confirmed. The beet leafhopper, Circulifer tenellus, was first confirmed to carry the S. citri pathogen. Later, S. citri was shown to be carried by two other species of leafhopper, Scaphytopius nitrides and S. acutus delongi, as well as several other species of insects.

In addition to citrus, various other plants have been shown experimentally to be hosts of S. citri; however, many of these experimental hosts do not appear to be hosts of S. citri in natural conditions. Some of the most important alternate hosts include various members of the Brassicaceae (mustard family), which are quite common in California as weeds, native species, or crops. Brassicaceae species are also hosts of the beet leafhopper, C. tenellus, the most important vector of stubborn disease in California despite citrus not being its preferred host. Most of the Brassicaceae that host S. citri are winter annuals and harbor the pathogen during the winter months. It appears that during the spring and early summer, C. tenellus migrates from the alternate Brassicaceae hosts, which are found in the foothill areas surrounding citrus production in the San Joaquin Valley, to the valley floor. The insects remain active as the season progresses and conditions for transmission remain suitable. As mentioned previously, it takes several months for initial infections of S. citri to build up in the plant. In the fall, C. tenellus migrates away from the valley floor towards the foothills. The foothills stay warmer than the valley floor during the winter months, and so provide a more suitable temperature for both C. tenellus and S. citri. So, as titer of S. citri decreases during the winter months, its perpetuation is assured by its presence in the foothills. Under experimental conditions, S. citri maintained in planta long-term has been demonstrated to lose its ability to be transmitted and apparently S. citri requires passage through the insect vector to retain its infectivity.

This implies that there would be little citrus-to-citrus transmission of stubborn by the leafhopper vectors under most circumstances, with the exception of young plantings. The spread of S. citri into citrus plantings apparently mostly comes from the alternate hosts in surrounding fields. Elimination of the pathogen from a grove or nursery would not prevent infection from an inoculum pool in the alternate hosts. The fact that the disease can apparently overwinter in areas removed from commercial production (ie, the foothills around the valley) and then be transported by the vector to the production area during the spring months means that elimination of alternate hosts or the vector from near the orchard would reduce but not eliminate the possibility of infection.

The much lower apparent susceptibility of older trees to infection is an advantage from the disease management standpoint. Orchards established with S. citri­-free trees in areas in which the populations of the beet leafhopper and the inoculum source in alternate hosts are consistently low for several years after establishment will greatly reduce the chances of stubborn infection and economic losses. The use of S. citri-free materials in areas with high S. citri presence in alternate hosts and high beet leafhopper activity would probably be of little use in the prevention of stubborn disease during years favorable to disease development. However, the use of pathogen-tested propagative material remains the first line of defense against all economically damaging citrus diseases. Maintenance of mother trees and/or production of young trees under screen provides protection against contamination with stubborn and other insect-vectored diseases and is a recommended practice.

Trap plants have in the past shown some usefulness in managing stubborn. Sugar beets are an attractant for the beet leafhopper but a non-host plant for S. citri. Sugar beets planted around a seedling planting of citrus reduced incidence of S. citri in periwinkle (an alternate host of S. citri) planted around the citrus seedlings by over 50 %. Although this did not provide complete control, the incidence was reduced enough that this strategy deserves consideration. Periwinkles planted around citrus would also provide an indication of the amount of S citri present as the periwinkle yellows symptoms develop. Vector control via the use of systemic insecticides has not been shown to be useful in the past in reducing the incidence of stubborn in either citrus seedlings or periwinkles. Antibiotics are effective against S. citri in vitro but apparently antibiotics injected into the xylem of large trees are not translocated into the phloem sieve tube elements in sufficient amounts for affected trees to improve.

Removal of stubborn-infected trees or branches is often practiced by growers. This is most effective in younger plantings, as it is at this stage that the trees are most susceptible to infection. Trees infected early in their life will never be productive, so, stubborn-infected trees in plantings less than 6 years old should be removed as soon as they are diagnosed. Trees that become infected after they are mature present only a small hazard to the rest of the grove since there is little tree-to-tree spread. It is mainly economic factors that will determine whether mature trees should be removed. Control of alternate hosts near these plantings will reduce (but not eliminate) the possibility of infection of re-planted trees.

In summary, the following are considered to be useful in lowering the incidence of stubborn or minimizing losses from it:

1. If possible, locate nurseries in areas where stubborn does not spread naturally and where the incidence of stubborn is low.

2. Use S. citri-free budwood for all propagations including topworking.

3. If possible, maintain mother trees under screen and if possible produce nursery trees in a protected environment (greenhouse or screenhouse).

4. Topworking should only be done on trees that are totally free of S citri.

5. On an annual basis, remove all stubborn-infected trees from orchards less than 6 years old.

6. All replants in orchards of any age should be removed if infected with stubborn and again replanted with stubborn-free trees.

7. Maintain a strict program of weed control in and around the planted orchards, particularly for the first 6 years.

8. In nurseries located in areas where S citri is endemic, substantial borders of attractive trap plants that are not hosts to S. citri can be used. The traps plants can also be treated with insecticides to kill the attracted leafhoppers.

9. Avoid the use of cover crops susceptible to S. citri in orchards less than 6 years old in areas with high populations of the insect vectors.

Infection by the stubborn disease pathogen, Spiroplasma citri, has caused the mature citrus fruit in the foreground to remain green, color unevenly, and remain smaller than normal.