Overview of Rice Yellow Mottle Virus

Overview of Rice yellow mottle virus

G. Konatéa and D. Fargetteb

aInstitut national pour l’étude et la recherche agronomique (INERA), Democratic Republic of Congo bIRD, UR “Resistance des plantes” BP 5045 34032, Montpellier Cedex 1, France

Abstract The rice yellow mottle disease is the main virus disease found on rice in Africa. It has been reported in all the major rice producing zones of sub-Saharan Africa (East, West, Central and Southern Africa, and Madagascar). The disease incidence and accompanying yield losses could be high depending on the rice cultivars grown, the date of infection, and type of rice. The Offi ce du Niger in Mali reported 80% incidence and more than 50% crop losses in 1995. RYMV is confi ned to the African continent. The Rice yellow mottle virus disease (RYMV) is caused by a Sobemovirus transmitted by beetles. The fi ne structure of RYMV has been recently described, thanks to electron microscopy and x-ray diffraction. Thus, it has been shown that some of the 180 identical subunits forming the viral coat were closely interlinked through an exchange of the internal part of the coat, which makes the virion particularly stable. Serological (monoclonal antibodies) and molecular (sequencing and phylogenic analysis) studies of RYMV led to the characterization of several serotypes and phylogenic groups with a well-defi ned, geographic basis. The pathogenicity study of RYMV strains made it possible to identify at least two aggressivity levels: common aggressivity responsible for severe symptoms found on all the varieties tested so far, except for a few cultivars belonging to the O. glaberrima species, and severe aggressivity that can circumvent most forms of resistance to the previous one. Two types of resistance to RYMV were identifi ed in rice (Oryza sp.): a mild polygenic resistance in Oryza sativa, japonica type which can slow down the spread of the virus and a high monogenic resistance in O. glaberrima which prevents it. These two types of resistance were harnessed in the development of virus-resistant varieties. Transgenic rice varieties in which the gene from the virus coat protein has been inserted were also developed but did not show any resistance to the virus. On the other hand, the insertion of all or part of ORF2 gene conferred to rice a level of resistance as high as that of natural resistance. Epidemiological studies of RYMV showed that the virus is not rice seedborne and that its natural hosts are very few. So the primary source of the virus inoculum is not known for a certainty. However, recent work conducted in West Africa put the blame

1 Plant virology in sub-Saharan Africa on cultivated rice regrowths and wild rice. On inspection of on-farm rice nurseries, cultivated rice regrown plants of Oryza longistaminata, and some rice seedlings were identifi ed with RYMV infection. And yet the same research work indicated that an epidemic could be initiated by transplanting a nursery with only 2.5% of infected seedlings. How the virus spreads in the fi eld is still an enigma. As a matter of fact, no link could be established between: (a) the spatial distribution of the disease and the transmission of the virus by beetles; (b) population densities of the vectors and disease incidence; and (c) cropping activities and on-fi eld propagation of the virus. Work on RYMV relied on the most state-of-the-art tools of serology, molecular biology, genetics, and biophysics. Thus a large amount of sometimes original data could be gathered on the virion. Conversely, our knowledge about rice mottle disease is still relatively limited. Future research should focus on the following themes: (a) the ecol- ogy of RYMV and identifi cation of primary inoculum sources; (b) characterization of primary modes of infection and on-fi eld propagation of the virus; and (c) development of integrated pest management strategies to combat the virus.

Résumé La panachure jaune du riz est la principale maladie virale du riz en Afrique. Elle a été rapportée dans toutes les grandes régions au sud du Sahara (Afrique orientale, occiden- tale, centrale, australe et Madagascar). L’incidence de la maladie ainsi que les pertes de production peuvent être considérables, dépendant des cultivars de riz, de la date de l’infection et du type de riziculture. A l’offi ce du Niger au Mali, des incidences de 80% et des pertes de production de plus de 50% ont été rapportées en 1995. La panachure jaune du riz est confi née au seul continent africain. Le virus responsable de la maladie, le Rice yellow mottle virus (RYMV), est un Sobemovirus transmis par des coléoptères. La structure fi ne du RYMV a été récemment décrite grâce à la microscopie électronique et la diffraction par rayons X. Ainsi, il a été démontré que certaines des 180 sous-unités identiques qui constituaient la capside du virus étaient étroitement associées les unes aux autres grâce à un échange de la partie interne de la capside, assurant une stabilité particulière au virion. L’analyse sérologique (anticorps monoclonaux) et moléculaire (séquençage et analyse phylogénique) du RYMV ont permis de caractériser plusieurs sérotypes et groupes phylogéniques ayant une base géographique marquée. L’étude de la pathogénicité des souches du RYMV a permis de caractériser au moins deux niveaux d’agressivité : une agressivité com- mune responsable de symptômes intenses sur toutes les variétés éprouvées à ce jour sauf quelques cultivars appartenant à l’espèce O. glaberrima et une agressivité sévère capable de contourner la plupart des résistances à la précédente.

2 Overview of Rice yellow mottle virus

Deux types de résistance au RYMV ont été identiﬁ és chez le riz (Oryza sp.). Une résistance modérée polygénique chez Oryza sativa type japonica capable de ralentir la propagation du virus, et une résistance élevée monogénique chez O. glaberima empêchant cette propagation. Ces deux types de résistance ont été exploités pour créer des variétés de riz résistantes au virus. Des variétés de riz transgéniques chez lesquelles le gène de la protéine de capside du virus a été inséré ont également été créées, mais elles n’ont pas présenté de résistance au virus. Par contre, l’insertion de tout ou partie du gène ORF2 a conféré au riz un degré de résistance aussi élevé que celui de la résistance naturelle élevée. Les travaux sur l’épidémiologie du RYMV ont montré que le virus n’était pas transmissible par les graines de riz et qu’il avait très peu d’hôtes naturels. La source d’inoculum primaire du virus n’est donc pas connue avec certitude. Cependant, des travaux récents en Afrique de l’Ouest ont incriminé les repousses de riz cultivé et le riz sauvage. En effet, l’inspection de pépinières de riz installées dans les champs a mis en évidence la présence de plants de repousses de riz cultivé, d’Oryza longistaminata et de plantules de riz infectés par le RYMV. Or, il a été montré par les mêmes travaux qu’il était possible de provoquer une épidémie en repiquant une pépinière où seule- ment 2,5% de plantules étaient infectés. Le mode de propagation du virus au champ reste une énigme. En effet, on n’a pas pu établir de relation entre : (a) la distribution spatiale de la maladie et la transmission du virus par les coléoptères ; (b) les niveaux de populations de ces vecteurs et l’incidence de la maladie ; (c) les opérations culturales et la propagation du virus au champ. Les travaux réalisés sur le RYMV ont utilisé les outils les plus modernes de la sérolo- gie, de la biologie moléculaire, de la génétique, et de la biophysique. Ils ont permis d’accumuler une foule d’informations quelques fois inédites sur la connaissance du virion. A l’inverse, notre connaissance des épidémies de panachure reste relativement limitée. Les recherches futures devraient être axées sur les points suivants : (a) l’écologie du RYMV et l’identiﬁ cation des sources d’inoculum primaire ; (b) la caractérisation des modes de contamination primaire et de propagation du virus au champ ; (c) le développement de méthodes intégrées de contrôle du virus.

Introduction The Rice yellow mottle virus disease was for the ﬁ rst time observed in 1966 in Kenya, East Africa, by Bakker. The disease is caused by a Sobemovirus of about 28 nm (Bakker 1974; Seghal 1981; Hull 1988). In the quarter of a century which followed its discovery, RYMV was not granted any particular attention by research owing to its low economic importance. The virus is now found in all sub-Saharan African countries and in Madagascar in the Indian Ocean where it is responsible for the most severe rice

3 Plant virology in sub-Saharan Africa disease (Raymundo and Buddenhagen 1976; Fauquet and Thouvenel 1977; John et al. 1985; Fomba 1988; Konaté 1995; Konaté et al. 1997). Yield losses could vary from 25 to 100% depending on how early the infection sets in, the type of rice cultivation, and the rice cultivars used (Taylor et al. 1990; Awoderu 1991; Konaté et al. 1997). West Africa has been confronted with highly devastating epidemics of RYMV disease since the early 1990s. The reasons for such epidemics appear to be linked to intensive rice cultivation due to increasing adoption of new production modes such as water-fed rice farming, annual double cropping, and high yielding but highly susceptible to RYMV Asian varieties. Faced with the serious threat from RYMV, virologists in Africa, Amer- ica, and Europe joined hands to develop new control strategies against the virus. This overview takes stock of our knowledge about the virus and proposes some guidance for future research work.

Structure of RYMV virus particle The morphology, size and composition of the RYMV virus particle have been described by Bakker (1974); the particle has a bear and isometrical nucleocapsid with a diameter of about 28 nm. It is made up of 23% nucleic acid and 17% protein. The nucleic acid which forms the virus genome is a single-stranded 1.4 x 106 molar RNA (Bakker 1974). The fi ne structure of the RYMV capsid was recently identifi ed (Opalka et al. 2000; Qu et al. 2000). One could establish that the capsid was composed of 180 identical capsid subunits. Besides, a close relationship between some subunits was highlighted, thanks to an exchange of the internal part of the capsid. This so far unknown form of linkage between the components of the capsid could be the main cause of the stability specifi cally observed with the virus particle. The sequence and pattern of RYMV’s genomic RNA were determined by Ngon et al. (1994). RYMV’s genome is composed of a single-stranded positive RNA. The 5’ end is in a covalent linkage with a VPg protein and the 3’end is not poly (A) (Sehgal 1981; Francki et al. 1985; Hull 1988). The 4450 nucleotides forming the RNA are organized into four ORFs (Fig. 1) which overlap (with the exception of ORF1) and code for four proteins (Ngon et al. 1994): (1) the ORF1, extending from nucleotide 80 to nucleotide 553 codes for a protein with 157 aminoacids and a size of 17.8 K. It has been said to play an important part in the infection of the plant and to be involved in the propagation of RYMV in the rice plant (Bonneau et al. 1998); (2) ORF2, from nucleotide 608 to nucleotide 3607, codes for a polyprotein with 999 aminoacids and a size of 110.7 K. The 134 fi rst aminoacids of this polyprotein would correspond to the VPg protein, followed by the virus protease, the helicase and the RNA-dependent RNA-polymerase; (3) ORF3; located inside ORF2 and extending from nucleotide 2092 to nucleotide 2467, codes for a 126 aminoacid-polypeptide of 13.7 K whose function is not known and (4);

4 Overview of Rice yellow mottle virus

608 3607 4450

VPg ORF2 5' 3' ORF1 ORF3 ORF4

80 553 2092 2469 3447 4166

Figure 1.

ORF4, from nucleotide 3447 to nucleotide 4166, thus overlapping the 3’ end of ORF2, codes for a 26K protein which corresponds to the RYMV coat protein. The RYMV’s genomic RNA is very similar to that of SBMV, a typical member of the sobemoviruses (Ngon A Ayassi et al. 1994), but is more or less distinct from that of CfMV, another member of the group (Mäkinen et al. 1995).

RYMV epidemiology The distribution of infection by RYMV in rice fi elds varies considerably across the same region, but also across the same fi eld. Over relatively short distances of 3 to 10 m, fi elds completely infected by the virus could be observed alongside almost virus-free farms. Likewise, within the same fi eld, at least four patterns of distribution of infected rice plants could be observed as follows: (a) a few isolated plants; (b) small patches of infected plants; (c) many large land areas with diseased plants; and (d) fi eld with almost 100% infection. Such patterns of distribution of RYMV infection are yet to be explained. They could involve several types of transmission and propagation and as a result, several infectious sources.

Transmission of RYMV The two types of RYMV’s transmission now clearly admitted are: insect-borne transmission and transmission by artiﬁ cial mechanical inoculation. Bakker (1974) reported in East Africa several leaf beetle species with the potential to transmit the virus, namely Sesselia pussilla (Galerucinae), Chaetocnema pulla Chapius (Halticinea), C. dicladispa (Chrysispa) Kraatz (Hispinae), D. bayoni Gest (Hispinae), and Trichispa sericea Guerin (Hispinae). Hispa (Dicladispa) gestroi Chapius (Hispinae) was also reported by Reckhaus and Andriamasintseheno (1995) as a vector of the virus in Madagascar. However, the role of these vectors in rice yellow mottle disease epidemics is not well established. In East Africa, Bakker (1974) reported that the ﬁ rst diseased

5 Plant virology in sub-Saharan Africa rice plants were consistently found at the borders of rice plots along irrigation bunds, which suggests transmission of the virus by insect vectors from external sources. At the Offi ce du Niger, in Mali, Peters (1996) found RYMV completely infected rice farms by the side of virus-free farms, which suggests that the vectors play a minor role in the spread of the virus. In Côte d’Ivoire, one could not establish a correlation between Trichispa sericea population levels and disease incidence (Heinrichs et al. 1997). It was also formally established that RYMV’s infection is not seedborne (Bakker 1974; Fauquet and Thouvenel 1977; Konaté et al. 2001) even though the virus was detected in all the rice seed parts (lemma, caryopsis, and embryo) (Abo 1998; Konaté et al. 2001). It is generally admitted that viruses found in the embryo are seedborne. As for RYMV, Konaté et al. (2001) have shown that absence of transmission was due to the inactivation of the virus during the maturation stage of the rice seed. Such inactivation phenomenon contradicts the results obtained by Abo (1998) stating that empty spikelets and aboveground contaminants of hulled rice seed were infectious RYMV sources. Several other RYMV modes of transmission have been suspected and more or less verifi ed. Thus, despite the abundant rice nematode fauna, none of the nematode species tested had been able to transmit RYMV (Bakker 1974; Raymundo et al. 1979; Abo 1998). Abiotic transmission of RYMV was strongly suspected but is yet to be demonstrated. According to Reckhaus and Andriamasintseheno (1995), transplanting rice into a soil containing cow dung and poorly decomposed crop residues could be responsible for the mechanical transmission of RYMV. Similar types of transmission could be observed when rice is transplanted into a soil on which infected rice regrowths and roots have been manipulated during plowing operations (Abo 1998). RYMV might have been also transmitted mechanically by contact of the gustation liquid and irrigation water with rice plants (Bakker 1974 or 1985). The role of men in RYMV mechanical transmission is highly suspected, mainly during cropping operations such as transplanting, fertilizer application, irrigation, harvesting, etc. Other assumptions point to contact between infected agricultural implements and rice plants, between contaminated hands and rice plants, and between healthy and diseased plants under the effect of wind, men, or animals (Abo 1998). Infection of rice farms from nurseries infected through contact between diseased and healthy plantlets or between contaminated hands and rice plants was suspected as far back as 1974 by Bakker, but he was not able to demonstrate it. The observation where RYMV is always more severe in farms with transplanted rice than in farms with direct sowing tends to confi rm such a hypothesis (Sy 1994). Traoré and Traoré (2000) showed that about 20% of nurseries at Sélingué in Mali were infected by RYMV at rates varying between 0.08 and 0.44% with a mean of 0.30%. They could

6 Overview of Rice yellow mottle virus also observe infected rice regrowths at a density of around 1plant/m2 in the nurseries. Trials conducted in Kamboinsé revealed that a 2.5 infection of nurseries is enough for an outbreak of RYMV (Fig. 2). From an epidemiological viewpoint, the mode of RYMV infection of the ﬁ rst rice plant (primary) and its mode of propagation to other rice plants (secondary) are yet to be determined even though several natural sources of the virus have been identiﬁ ed.

RYMV natural sources Contrary to most other plant pathogens, viruses are absolute parasites; they can survive only within their hosts. Plant viruses in particular will ﬁ nd other hosts when the true host is absent during a certain period of the year. These may be seeds, roots or tubers, alternative hosts, vectors, crop residues, animal waste, lake or river water, etc. As for RYMV, several investigations have been carried out in order to identify their sources of survival as well as their role in Rice yellow mottle virus epidemics.

RYMV alternative hosts In East Africa, Bakker (1974) showed that the following Poacea species could be experimentally infected by RYMV and, therefore, are likely to host the virus: Dine- bra retroﬂ exa (Vahl) Panz., Diplachne caudata K. Schum., Eragrostis aethiopica Chiov., Eragrostis ciliaris (L.) R. Br., Eragrostis namaquensis Nees var. namaquensis,

,QFLGHQFH5<09

-RXUVDSUqVUHSLTXDJHDays after transplanting

Figure 2.

7 Plant virology in sub-Saharan Africa

Eragrostis tenella (L.) Reom. Et Schult., Oryza barthii A. Chev., and Oryza punctata Steud. However, rice is the only host found with natural infection. In West Africa, Konaté et al. (1997) showed that Echinochloa colona, Ischaemum rugosum, Panicum repens L., Oryza longistaminata A. Chev. Et Roehr., and O. barthii A. Chev. were naturally infected by RYMV. In Central Africa, Traoré (2000) reported natural infection on Oryza barthii, O. longistaminata, Panicum subalbidum Kunth, Acroceras zizanioides, Sac- ciolepis africana, Eragrostis sp, and Setaria longiseta in Cameroon and Chad. With regrowths, rice seems to offer the best source of preservation to the virus. Konaté (1995) reported that in Burkina Faso, infection rates of 15–20% were recorded on rice regrowths throughout the dry season. The regrowths could survive thanks to irrigation and double cropping.

Rice seed Bakker (1974) and Fauquet and Thouvenel (1977) showed that RYMV could not be transmitted by the rice seed. However, Abo (1998) could detect the virus in the lemma but not in the embryo. Konaté et al. (2001) subsequently proved that RYMV was present not only in the lemma but also in the caryopsis and the embryo and still was not seedborne. Investigating virus infectivity during seed maturation, they were able to ﬁ nd almost 100% loss of RYMV-infectious potential at maturity (Fig. 3). The cause of the inactivation of the virus during the maturation phase of the seed is being investigated. The results now seem ﬁ nal that rice seed is not a survival source for RYMV.

HFWLRQ I ,Q

, ,, ,,, ,9 9

Figure 3. 6WDGHGHIRUPDWLRQGHODStages of grainJUDLQH formation

8 Overview of Rice yellow mottle virus

Other means for RYMV preservation Several media have been suspected to host RYMV; irrigation water (Bakker 1974), crop residues (Abo 1998), and animal waste (Reckhaus and Adriamasintseheno 1995). Increasing use of cow dung as organic manure in rice cultivation prompted our team to look for RYMV in such an environment. It has been established that when a cow is fed with RYMV-infected rice straw, the infectious virus can be easily detected in the fresh dung using ELISA test. However, the virus is fast degraded and loses its infectious potential when the cow dung is left to dry in the conditions of the pen; it cannot be detected after 2–3 weeks (Sarra 1998) (Fig. 4). Consequently, cow dung is not a favorable environment for RYMV preservation. Sarra (1998) reported that RYMV would remain infectious in rice straw 10 months after harvest, and roughly 6 months after harvest in rice roots sampled in the soil during the dry season.

RYMV biodiversity Over the last ﬁ ve years, RYMV’s variability is been actively investigated at serological, molecular, and pathogenic levels in support of efforts aimed at the development of genetic

2.5 Infected straw Uninfected straw

2.0

1.5

1.0 Absorbant at 405 nm 0.5

0.0

0 1 234 5 6

Figure 4. Time after sampling (weeks)

9 Plant virology in sub-Saharan Africa control measures against the virus. The use of polyclonal and monoclonal antibodies has made it possible to characterize ﬁ ve RYMV serotypes (three in West and Central Africa and two in East Africa and Madagascar) that are adapted to different geographic zones (Konaté et al. 1997; N’Guessan et al. 2000) (Fig. 5). The comparison of coat protein sequence of isolates from those serotypes showed that the three serotypes from West and Central Africa corresponded to three distinct strains and two main phylogenic groups. The serotype from East Africa corrolated with two strains and two philogenic

Rice ﬁ eld

- Primary infection s s i

- Secondary infection s

e M

Breeding s

t ground

t Primary infection

n I n

o i

s s i

Insect vectors r o m t s

Mechanical transmission c n e a v r t t c l e a s ic In n a h c e M

Environment

Figure 5. A descriptive model of Rice yellow mottle virus (RYMV) epidemiology. Mechanical transmission may result from: (1) cultural interferences by man such as transplanting, weeding, fertilizer spraying, irrigation, harvest; (2) animal interferences through grazing; (3) animals that are rice pests; (4) wind.

RYMV primary inoculum source - Regrowth of infected rice - Natural hosts of RYMV (wild rice and Poaceae) - Cultural rubbish (roots and straw of infected rice)

10 Overview of Rice yellow mottle virus groups, while that of Madagascar corresponded to one strain and one phylogenic group (Pinel et al. 2000; Traoré, 2001) (Fig. 5). Serological and morphological variation of RYMV has not yet been clearly linked up with any pathogenic variation of the virus. One of the three strains from West and Central Africa conﬁ ned to the agroecological zone extending between 600 and 800 mm rainfall proved particularly aggressive in infecting rice cultivars so far considered highly resistant, and even immune, namely TOg5681 and Gigante (Konaté et al. 1997). N’Guessan et al. (2000) reported in Côte d’Ivoire the domination, under mixed infection, of the strain from the northern Guinea savanna by the one from the forest zone, both in terms of coat protein and RNA accumulation. Nevertheless, they showed that such domination was not due to a difference in aggressivity between the two strains in relation to most of the rice cultivars tested. Considerable research efforts should be made to characterize the pathogenicity of RYMV variants and to determine their role in rice yellow mottle epidemics.

Interactions between RYMV and Oryza sp. The degree of susceptibility of the host to the pathogen plays a critical part in the outbreak of epidemics. Both in men and animals, immunization controls epidemics by reducing the level of susceptibility. In plants, genetic resistance is exploited. In the case of RYMV, a lot of research work was carried out in order to identify sources of resistance and to understand the genetic factors governing such resistance (Attere and Fatokun 1983; Okioma and Sarkarung 1983; John et al. 1985; John et al. 1986; Fomba 1988; Thottappilly and Rossel 1993; Paul et al. 1995; Albar et al. 1998; Coulibaly et al. 1999; Ioannidou et al. 2000). Many and often contradictory results were generated, most probably because of the use of different virus isolates and infection conditions. However, it has been consistantly shown that almost all sources of resistance to RYMV belong to Oryza glaberrima which originated from Africa. Two types of recessive resistance were identiﬁ ed: a partial polygenic resistance in Oryza sativa japonica (upland rice) and a high monogenic resistance in O. glaberrima (Ndjiondjop et al. 1999). Recessive resistance of rice to RYMV and fertility barriers between O. glaberrima and O. sativa have so far constituted the major hindrance to the development of resistant rice varieties using conventional breeding methods. To circumvent this problem, the transfer of all or part of the virus gene has been attempted with relative success. The transfer of the gene coding for the virus coat protein did not confer any resistance to rice. On the other hand, that of all or part of ORF2 made it possible to obtain varieties with a level of resistance as high as that of cultivars with a high natural resistance (Pinto et al. 1999). The exploitation of such resistance could become a major component in RYMV control.

11 Plant virology in sub-Saharan Africa

Role of climatic factors in outbreaks of RYMV disease Climatic factors such as rain, relative humidity, temperature, and sunshine play an important part in oubreaks of epidemics of virus diseases. They usually inﬂ uence viruses, vectors, main alternative virus hosts, etc. In the case of RYMV, very few data are available regarding the role of such climatic factors in the development of rice yellow mottle epidemics. Geographical adaptation of RYMV strains as reported by many authors is probably related to climatic factors which either directly impact on the virus, or do so through alternative hosts, the occurrence of which depends on such factors. While investigating the factors which inﬂ uence the development of the disease symptoms in Nairobi, Kenya, and in Wageningen, the Netherlands, Bakker (1974) observed that high temperatures (30 °C and above) would reduce the period for the expression of symptoms, and that low relative humidity would trigger the necrosis of infected rice leaves. Awoderu (1991) reported a variation of the incidence and severity of RYMV depending on climatic zones and the local environment in which rice is grown (type of rice cultivation). For example, with irrigated rice and in the Sahel, the disease incidence and severity are higher than in the tropical rain forest where they vary from low to moderate regardless of the type of rice cultivation. Given that climatic zones are the result of a combined effect of the different climatic factors, the individual impact of each factor is still unknown.

Epidemiological model of RYMV disease Relevant information on RYMV’s epidemiology is scanty. However, factors such as vector transmission, existence of natural sources of effective preservation as well as the numerous assumptions on abiotic transmission modes of the virus could be used to develop an epidemiological model (Fig. 5). The model would function this way: (1) RYMV survival kit made up of infected rice regrowths, the virus Poaceae hosts, and crop residues will form the primary source of inoculum; (2) direct infection of new rice farms may emanate from that source through RYMV transmission by insect vectors or mechanical transmission by men; (3) rice nurseries can also be infected from that source mainly through transmission of the virus by insect vectors; (4) the new rice farms planted with materials from those nurseries are basically infected by men during transplanting operations; and (5) the spread of the virus in those ﬁ elds is caused by men, animals, wind, and irrigation water.

RYMV multiplication and migration in the rice plant The site of RYMV was observed for the ﬁ rst time in leaf tissues of infected rice plants by Bakker (1974). He showed that the virus was present in the cells of the epidermis,

12 Overview of Rice yellow mottle virus mesophyll, and stomata. On the other hand, no virus particle could be observed in the chloroplasts and the cell nucleus. Opalka et al. (1998) and Ioannidou et al. (2000) reported a strong RYMV build-up in the xylem cells. RYMV migration was thought to take place mainly from this tissue via the plasmodesmes for short-distance (cell to cell) migration and sap vessels for long-distance migration.

RYMV diagnosis The observation of RYMV symptoms as a means of diagnosis quickly showed its limitations as many other stresses such as mineral defi ciencies can cause similar symptoms. RYMV is a virus which multiplies profusely in the rice plant and huge quantities of viral particles can be isolated by purifi cation (Bakker 1974; Fauquet et Thouvenel 1977). Furthermore, RYMV is highly immunogenous with no serological linkage with any known virus (Bakker 1974; Fauquet and Thouvenel 1977). The immunoserological methods were therefore quite appropriate for diagnosing the virus. The technique developed by Outcherlony (1962) has been widely used (Bakker 1974; Fauquet and Thouvenel 1977; John and Thotappilly 1987; Ngon and Yassi 1989). This is a technique with limited effi ciency: it is more or less weak in terms of sensitivity and requires a relatively high quantity of antiserum. The ELISA (enzyme-linked immunosorbent assay) technique with several variants (Clark and Adams 1977; Koenig 1981; Bar Joseph and Salomon 1984) is today the most widely used. Its sensitivity depends on the variant used, the serum source as well as the virus isolate (Konaté et al. 1997). Sarra (1998) reported that the use of a mixture of gamma immunoglobulins against three RYMV serogroups in a biotin/streptavidin-ELISA procedure allowed for the detection of each of the serogroups with RYMV 0.1–1 ng /ml sensitivity. The use of monoclonal antibodies in ELISA enabled the detection and characterization of specifi c RYMV isolates (Konaté et al. 1997; N’Guessan et al. 2000).

RYMV control RYMV appears as the most difﬁ cult virus in sub-Saharan agriculture today. The virus is particularly stable and seems to survive in particularly harsh weather conditions. Unlike the other major viruses of the region, such as African cassava mosaic virus (ACMV), Maize streak virus (MSV), Groundnut rosette virus, (GRV, GRAV, Sat-RNA) or Tomato yellow leaf curl virus (ToYLV), all speciﬁ cally transmitted by vectors, RYMV is transmitted via several modes. In agronomically desirable rice varieties, there is virtually no source of sound resistance to RYMV. Although the pathogenic meaning of large biodiversity is not yet known, it seems it could help circumvent all the problems of

13 Plant virology in sub-Saharan Africa resistance experienced so far. In light of the situation described above, integrated pest management (IPM) seems to be one of the best strategies in combating the virus.

Future research on RYMV Our knowledge about the ﬁ ne structure and molecular biology of RYMV has tremendously improved over the last ﬁ ve years. On the other hand, our knowledge on the epidemiology of RYMV is limited. In order to improve the epidemiological data essential for the development of IPM methods, research efforts should be made with a view to: • Characterizing the pathogenicity of different RYMV strains. • Identifying primary sources of RYMV inoculum. • Determining primary modes of infection of rice in nurseries and farms. • Determining the modes of propagation of the virus in rice farms.

Conclusion Viewed for almost a quarter of a century after its discovery in 1974 as a minor rice infection, Rice yellow mottle virus has become, over the last ten years or so, the main threat to rice cultivation in Africa and Madagascar. Research efforts focused on the virus have been much more successful in the laboratory than in the ﬁ eld. Results turned out by laboratories have dramatically increased what is known about the virus at structural and molecular levels, but there are still a lot of gaps with respect to epidemiological knowledge. It is probably the reason why research is yet to come up with efﬁ cient methods to control the virus. References

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