TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.1 DETECTION/Symptomatology

Section 2.1.2Diseases Caused byPhytoplasmas in Potato

Many plant diseases that were presumed to be caused by viruses arereally associated with other kinds of pathogenic microorganismssomewhat resembling bacteria and rickettsia.

These plant pathogens are the Phytoplasmas (previously known asMycoplasma-like organisms or MLOs). They are transmitted byleafhoppers or psyllids and are associated with diseases like witches'-broom and yellowing.

P.V. • Sec 2.1.2 – 99 • Page 2 - INTERNATIONAL POTATO CENTER

Characteristics of phytoplasma organism:

• Pleomorphic microorganisms (0.1 to 1.0 �m in diameter) that canpass through filters with 450-nm pore diameter.

• Cells with a thick three-layer unit membrane.

• Ribosomes similar to those in bacteria.

• Contain DNA and RNA.

• Multiply by binary division.

• Some species can be cultured in artificial media. In agar, theyproduce typical tiny colonies resembling a fried egg.

• Penicillin resistant and tetracycline sensitive. Unlike L-shapes frombacteria, they do not revert to bacterial shape when grown inpenicillin-deficient medium.

• Susceptible to infection by viruses.

About 40 plant diseases have been reported to be caused byphytoplasmas. Most of the evidences were obtained through electronmicroscopy. Since they are pleomorphic, it is difficult to describe theirshape, which varies according to the method used to prepare thesamples for electron microscopy. Microorganisms, which differ fromphytoplasma, have been observed in dwarf maize plants. They have aspiral shape and are about 250 nm wide and 12 �m long. These arecalled spiroplasmas.

Some phytoplasmas have been cultured in artificial media and shown toinduce disease in inoculated plants. These diseases are potato purpletop, sandle spike, mulberry dwarf, pear decline, etc., and are all of greateconomic importance. They infect the plant systematically and generallyhave a wide host range. Some symptoms produced in plants byphytoplasma are very characteristic and specific. They include witches’-broom, virescence (flowers turning green), and phyllody (petals changingto foliar structures).

Electron microscopic studies show that most phytoplasmas are restrictedto the phloem of the plant tissue. Although the phytoplasmas are largerthan the pores of the sieve cells, being pleomorphic, they can changetheir shape temporarily and pass through the pores to adjacent cells.

It has been reported that leafhoppers and psyllids are phytoplasmavectors. Many phytoplasmas also infect their vectors and affect their lifecycle. Normally, the vectors infected by phytoplasmas are never cured.Some are also transmitted by transovaric passage.

The phytoplasmas are susceptible to antibiotics, especially to thetetracycline group, but they are penicillin-resistant. When an infectedplant showing symptoms is periodically inoculated with a tetracyclinesolution, the symptoms normally disappear. In cases where thesymptoms are not evident, their occurrence is delayed. It is important tonotice that tetracycline does not cure the plant, since the symptoms re-appear after the tetracycline treatment.

P.V. • Sec 2.1.2 – 99 • Page 3 - INTERNATIONAL POTATO CENTER

The phytoplasmas are also sensitive to high temperatures. Hence, theycan be removed from infected plants, plant parts, and propagative organsduring dormancy using heat treatment (30-37ºC). This procedure couldtake weeks or months, depending on the phytoplasma involved.

L-shaped bacteriaWhen bacteria are cultured in a medium with penicillin, they are unable toproduce wall cells. These bacteria without walls are called L-shapes.They look very similar to phytoplasma but are only formed underlaboratory conditions and are very unstable: once penicillin is removedfrom the culture medium, they regain their original shape.

Diseases caused by phytoplasma in potato plantsIn 1915, Maurice discovered phytoplasma for the first time. Diseasescaused by phythoplasma in potato plants are common worldwide, but insome countries (such as Guatemala, Panama, Mexico, and Tunisia),they are as important as other diseases that affect crops and hence theeconomy of the country. These diseases have different names and thesymptoms are very different. As different microorganisms can producealmost identical symptoms in different potato cultivars and in differentplant species, symptomatology is not a very reliable characteristic,although very thorough, detailed descriptions have been reported byprevious researchers.

Despite the limitations of using symptomatology as a tool for diagnosis,certain symptoms are regularly associated with phytoplasma-likeinfections. According to these symptoms, the diseases caused byphytoplasma in potatoes can be classified in two groups: aster yellowsand witches'-broom.

1. Aster yellow groupThis group has different names, including purple top, purple top wilt,haywire, late-breaking virus, stolbur, parastolbur, metastolbur, tomatobigbud, and purple top roll. Although this group is found worldwide, inmany countries it is not regarded as economically important.

The symptoms vary considerably, suggesting great variability amongphytoplasma types. The severity of symptoms and the extent of theinfection to the tuber also varies, probably due to the occurrence ofdifferent strains of phytoplasmas. Since there are no appropriatediagnostic methods, these differences among phytoplasmas have notbeen studied. The symptoms have sometimes been mistaken for thoseproduced by Rhizoctonia or Fusarium infections as well as with theprimary symptoms of PLRV infections.

Normally, the symptoms do not show until flowering. The base of theyoung leaflets turns purple, red, or yellow. The petiole stands erect, theinternodes shorten, and the whole plant grows straight up. Chlorosis maybe generalized, and the leaves turn upward. Aerial tubers may be formedin the axillary buds. The vascular bundles lose color, in some cases veryseverely. The root neck rots, causing wilt. The stolons show browning,which can spread to nearby tubers. Some aster yellow strains prevent

P.V. • Sec 2.1.2 – 99 • Page 4 - INTERNATIONAL POTATO CENTER

infected tubers from sprouting or produce elongated sprouts that, in turn,produce weak plants that do not survive.

Another type of aster yellow is haywire, which is transmitted by seedtubers. Plants growing from these seed tubers show severe symptoms ofpurple top wilt, dwarf, curled leaves, and purple or red leafletsimmediately after plants emerge. The aster yellow phytoplasmas have awide host range that includes plants from different families. The vectorsare Macrosteles divisus and M. fascifrons.

Stolbur or parastolbur (reported in Europe) is very similar to aster yellow,but the symptoms are severe and can make the plant wilt and die soonafter they appear. Stolbur does not spread by tubers, since the infectedones rot before sprouting. The vectors are: Hyalesthes obsoletus,Euscelis plebejus, Aphrodes bicinctus, and Macrosteles laevis.

EpidemiologyThe vectors of aster yellows do not show preference for potato plants ashosts under normal conditions; they live and propagate in grass andweeds. Due to the wide range of phytoplasma hosts, many of theseplants can become infected. The infection of a potato field byphytoplasma is established through the migration of leafhoppers fromthese weeds. Normally the insect inoculates plants or groups of plantsdispersed throughout the field. There is no evidence to show that theseinfected plants are a source of inoculum for the propagation of thedisease. It has also been reported that adults as well as nymphs areincapable of acquiring the phytoplasma from potato plants. This is to saythat the transmission takes place when the leafhopper has acquired thephytoplasma, days or weeks before, from weeds or other plants. Hence,the crisis in phytoplasma epidemics in potato fields depends on theactivity of migratory leafhoppers and the presence of inoculum in nearby[non-potato] plants.

2. Witches'-broom groupThis is also called northern stolbur or dwarf shrub virosis and has beenreported in North America, Europe, and Asia. The symptoms are roundedleaves that later develop chlorosis, and the development of many axillarybuds in the base of the plant. The plant stands erect, and dwarfing is acommon characteristic. Some leaves can turn purple or red. If the plantsflower, the flowers will be green (virescence) or will adopt the shape ofleaves (phyllody).

Unlike aster yellows, the disease is transmitted by the tuber. The firstyear of infection, the infected tubers look normal. However, when planted,they produce plants that show symptoms. The tubers of the second yearof infection are small and produce elongated sprouts. These tubers lacka dormancy period.

The vectors of the disease are Ophla (Scleroracus) flavopictus, S.dasidus, and S. balli. In many parts of the world, the vector of the disease

P.V. • Sec 2.1.2 – 99 • Page 5 - INTERNATIONAL POTATO CENTER

is still unknown. The vectors are unable to acquire the phytoplasma frompotato plants to further propagate the disease.

Identifying the diseaseIn order to apply the most suitable control measures for phytoplasmas inpotato plants, it is important to establish which phytoplasmas areassociated with the symptomatology of the disease. Even though this isof little help in the diagnosis, the characteristic symptoms caused by thephytoplasmas described above could be used as preliminary steps.

The transmission of the phytoplasma agent from suspected potato plantscould be achieved by grafting them to other hosts. The use of vectors totransmit a phytoplasma from these potato plants is not possible, sincevectors are unable to acquire the phytoplasmas from them.

The following species have been reported to be alternative hosts forphytoplasma: Datura stramonium, D. metal, Lycopersicon esculentum,Cyphomandra betacea, Nicotiana tabacum, Medicago sativa, Melilotusalba and Trifolium repens. Special care should be taken whenphytoplasmas are intended to be transmitted from field potato plantsthrough grafting, since these plants are usually infected by otherpathogens, especially viruses. These viruses could infect many of thesehosts and could cause symptoms leading to an incorrect diagnosis.

Some serological techniques have been established to identify thepresence of virus after grafting. If any virus is infecting the alternativehost, then it will be necessary to graft part of the plant infected with aphytoplasma in another potato cultivar immune to the virus. Other hostplants, immune to virus but phytoplasma sensitive, could also be used.The aim of graft transmission is to isolate phytoplasmas to performfurther research.

The next step is to test its sensitivity to antibiotics. In this experiment, it isimportant to have many phytoplasma-infected plants and to have enoughcontrols to obtain reliable results. One group of plants should beinoculated weekly with tetracycline solution, another with penicillinsolution, and the control group with distilled water. It should be noted thata high concentration of antibiotics is toxic for the plants. Hence, threeconcentrations of antibiotics should be used (100, 500, and 1000 ppm).

Healthy plants should also be inoculated with the three concentrations ofantibiotics in order to detect their level of toxicity. If, in the experiment, theconcentration of antibiotics used is not toxic, then higher concentrationscould be substituted. If the phytoplasma are associated with the disease,the plants that received the tetracycline treatment will show a reduction inthe severity of the symptoms (as long as these symptoms are not evidentin the young tissue that will continue to grow). The plants that receivedthe penicillin treatment will not show any change in the severity of thesymptoms. If the weekly tetracycline treatment is stopped, the plants willgradually show the symptoms of the disease again. Therefore, thesensitivity of the agent to tetracycline is a good indicator of a phytoplasmaassociated with a disease.

P.V. • Sec 2.1.2 – 99 • Page 6 - INTERNATIONAL POTATO CENTER

Petioles and stems of diseased plants can be dyed with Diene's stain*and observed under a light microscope. Evidence of phytoplasmas will bethe presence of dark-blue granular structures in the phloem. The finaltest to detect phytoplasmas is by means of electron microscopy. Samplesare obtained from the main vein, petioles, or stems of infected plants.The samples are cut into 2–3 mm pieces and soaked in Karnowsky fixingsolution** (or in glutaraledhyde solution 2% in 0.01 M phosphate buffer,pH 7.0). The prepared sample can then be sent to any lab with facilitiesfor electron microscopy work (for example: The Virology Section,International Potato Center (CIP), P.O. Box 1558, Lima 12, Peru).

Figure 1. Cross-section of “olluco” phloem showing phytoplasmabodies

*Diene’s Stain:Methylone Blue 2.5 gAzure II 1.25 gMaltose 10 gSodium Carbonate 0.25 gComplete to 100 ml with distilled water

** Karnowsky Fixing Solution:Paraphormaldehyde Solution 8% (12 ml)Cacodylate buffer 0.1 M, containingCa Cl2 2H2O 0.1% (33 ml)Glutaraldhyde Solution 70% (2 ml