Pathology

SUGARCANE BACILLIFORM VIRUS AND OTHER DISEASES OF SUGARCANE IN MOROCCO

L.J.C. Autrey, A. Madrane, F.W. Hesse and M. Nadi f

Centre Technique d e l a Canne b Sucre,

Kenitra, Morocco

Key words: Morocco, sugarcane, diseases, fungi, SCMV, bacil l i form virus,

non parasitic, frost damage

ABSTRACT

A new mechanically transmissible, latent virus designated as sugarcane bacilliform (SCBV) and sugarcane mosaic virus strain D (SCMV-D) have been indentified. Research on their epidemiology, host range, vector relationship and varietal reaction have been initiat- ed. lmmunodiffusion tests, ELlSA and immuno electron microscopy have been applied for their diagnosis. Measures have been successfuIly adopted for the control of SCMV-D, which is no longer found in the field, whilst the exact distribution of SCBV and its eco- nomic importance have to be established. The three fungal diseases recorded (pokkah boeng, red rot and brown spot) are minor. A negligible level of what is probably red stripe disease has been observed. Among the five non-parasitic diseases identified, leaf fleck, interveinal chlorosis, bunch top and variegation are minor while frost can cause considerable damage. An elaborate quarantine system has been established out- side cane growing areas and a four stage nursery system provides for the supply of disease-free material. Field tests for evaluating varietal reaction to smut and SCMV-D have been successfully carried out in Senegal and Spain respectively.

INTRODUCTION

Sugarcane was probably introduced to Morocco during the ninth century, and was for years one of the main supports of the economy through export to various European coun- tries (Schmidt et aPO). Sugar production declined rapidly in the seventeenth century, and soon ceased due to competition in the European market from colonies in the New World. However, the effect of an unidentified disease or pest in the decline has not been excluded (Berthier"). Sugarcane has been introduced from South Africa, Spain, Argentina, India, Java and the Caribbean zone on several occasions during the twentieth century. Grillot13 reviewed the trials carried out from 1915-1919. The crop did not become established between 1915 and 1960 owing to various constraints, mainly lack of irrigation, infestation with Sesa- mia spp and frost damage (Schmidt et aPO). The introduction of 2 000 cuttings of varieties Co 290, CP 44-101 and NCo310 in 1962 from Spain, and subsequent multiplication and experimentation by the lnstitut Nation- al de la Recherche Agronomique (INRA) in collaboration with the German Agency for Technical Cooperation (GTZ), allowed the industry to develop dramatically (Schmidt et

afO). Research is carried out at present by INRA and the Centre Technique de la Canne a Sucre (CTCAS), the latter benefitting from technical support from GTZ. Sugarcane is cultivated on approximately 17500 ha in three coastal districts in the north of Morocco. These are Gharb and Loukkos on the Atlantic, and Basse Moulouya on the Mediterranean. Sugar production amounted to 115 000 tons in 1988. Major varieties in- clude CP 44-101, CP 65-357, L 60-25 and L 62-96, with average cane yields of 77,6 and 68.1 tons cane per hectare in Gharb and Loukkos, the maior areas.

Various diseases and pests have been identified although it would appear that, except for frost damage (Schmidt et all8, Schmidt et all9) and sugar cane mosaic virus (Fisher and LockhartI2), significant losses have not been sustained by the industry. The phytosan~tary situation has been described in various reports by Barat (unpublished 1969), Autrey2,3,4,5,6,7, Autrey and Madrane8, Baudin9, BendaIo, Schmidt ef aPO.

PARASITIC DISEASES OF SUGARCANE

Virus Diseases

Two virus diseases have been identified in Morocco: sugarcane bacilliform (SCBV) and sugarcane mosaic (SCMV). Virus particles with bacilliform morphology 131 + 7 nm X 31 + 2 nm, which had not previously been reported in sugarcane, were found during electron microscope examinations of sap extracted from shoots of variety Mex 57-473 with abnormal mosaic symptoms (Autrey3). Further work showed that virus particles were also present in shoots of the same variety without any visible internal or external disease symptoms, and growing in various field trials and multiplication plots in Gharb (Lockhart and AutreyI5). Preparation of antisera facilitated greatly the detection of the latent virus by ELlSA and immune electron microscope (IEM), as well as by less sophisti- cated techniques such as agar gel diffusion tests. Although SCBV was mechanically transmitted to sugarcane, it did not infect various other plants including banana, maize, barley, wheat, oats, johnson grass and three test plants as checked by ELLA and IEM. In direct ELlSA tests, SCBV was detected in plant sap dilut- ed two-fold and in partially purified and purified preparations. In further ELlSA tests, Autrey7 found that in plants coinfected with SCMV, a higher concentration of SCBV par- ticles was present (Fig 1) confirming similar observations made earlier with the electron microscope (Autrey3). The virus was also diagnosed in one out of 17 symptomless leaf

OD, 406 nm 0.8

u. u Buffer Healthy SCMV SCBV SCBV+SCMV

(--- Controls --->

Figure 1. Detection of sugarcane bacilliform virus (SCBV) by direct ELlSA in sap ex- tracts from plants with and without sugar cane mosaic virus (SCMV) infection

PATHOLOGY

samples of CP 44-101 taken at random in commercial fields in the Gharb area. SCBV was found to be serologically related to banana streak virus but not to six other similar small, non-enveloped bacilliform viruses.

The origin of SCBV in Morocco has not been established, but it may have been introduced c

through quarantine in 1979 in variety Mex 57-473, all plots of which were infected. The finding of the virus in Mex 57-473 in Hawaii (Lockhart and Autrey15) gives circumstan- tial supporting evidence.

I The economic importance of SCBV is not known at this stage, but it may be responsible for yield decline in Mex 57-473 according to data available from field trials. Its effect

I in sugarcane is being established. The presence of SCMV in Morocco was first suspected by Barat in 1969 (Baudin9). The presence of strain D of SCMV in fields of NCo310 was confirmed by Fisher and Lockhart12. Although infection was widespread in the 1 450 ha ~ l a n t e d with NCo310, losses were never assessed, although Fisher and LockhartI2 mentioned "increasing losses in the vari- ety': Indirect losses were induced by the rapid and premature ploughing out of fields of NCo310 and its total eradication by the end of 1983. The disease has since then been kept in a glasshouse well outside the cane growing areas. SCMV-D has never been observed in any other sugarcane variety or alternative host plants despite exhaustive surveys (Autrey3). It is thought that the virus was probably introduced through infected cuttings from Spain in 1962. Surveys in the Costa del Sol area from which the cuttings originated have revealed widespread infection in NCo310 (Autrey and Madrane8). Studies in Beltsville, USA on material sent from Spain and Morocco revealed that SCMV-D was present in both countries (Gillaspie, pers commun).

Owing to the potential economic importance of SCMV (Koike and Gillaspie14) and the possibility that the disease is present in alternative hosts or is latent in cane, a research project has been implemented to study various aspects of the disease (Autrey3p4r5). Commercial and promising varieties have been evaluated against SCMV-D under glass- house conditions since 1985. These revealed that commercial var~eties CP 65-357 and L 60-25 were susceptible to the disease (Autrey3), so vigilance is needed during bulk- ing up in the field. In the latest test (Autrey7), 11 varieties were symptomless and clas- sif~ed as highly resistant, while 13 others were assessed on the basis of percentage in- fection and severity of symptoms as resistant to highly susceptible. SCMV-D presence is diagnosed by direct and indirect ELlSA techniques with an antiserum to the Moroccan isolate. A rapid procedure for diagnosis by indirect ELlSA in six hours with the use of unclarified sap was developed (Autrey3), and this has detected SCMV-D in symptomless plants as well as those with good or doubtful symptoms. Field trials using NCo310 in spreader rows were established in Spain in 1985 and 1988 with commercial and promising varieties to evaluate their reaction to the virus ( A ~ t r e y ~ , ~ , ~ , ~ , Autrey and Madrane8 and Madrane et all6, Madrane et all7). The first tri- al showed low infection levels but tended to confirm the susceptible ratings obtained in glasshouse trials for varieties such as CP 65-357, L 60-25 and L 71-20. A second trial was established in spring 1988 with two experimental blocks, one with natural infection but in which maize had been planted to build up an aphid vector population, and the second in which the varieties had been inoculated mechanically. In the latter block level of infection was high reaching 57% in NCo310 and 75% in L 71-20 two months . after inoculation, while natural infection in blCo310 reached 24% six months after planting (Table 1). The field resistance of CP 44-101 was confirmed.

L.J.C. AUTREY, A. MADRANE, F.W. HESSE AND M. NADlF

Table 1 - Reaction of twelve varieties to SCMV-D following mechanical inoculation and exposure in the field at Estacion de Cartama, Spain.

Mechanical inoculation Exposure trial Variety

% Infection + Provision rating % infection ++ CP 44 101 0 Highly resistant 0 CP 70 321 0 Highly resistant 0 CP 72 355 0 Highly resistant 0 L 62 96 0 Highly resistant 0 L 72 85 1.1 Resistant 0 CP 61 37 17.7 Modertely resistant 0 CP 68 1067 33.4 Susceptible 9.2 CP 66 346 34.0 Susceptible 0 CP 72 1210 47.1 Highly susceptible 0.2 NCo 310 56.7 Very highly susceptible 23.8 CP 65 357 67.3 Very highly susceptible 17.6 L 71 20 75.1 Very highly susceptible 19.4

+ Mean of three replicates, two months after inoculation ++ Mean of three replicates, six months after planting

I it Bacterial diseases

( 1 Striping symptoms resembling closely those of red stripe Pseudomonas rubril~neans were t observed in nursery plots in varieties L 72-85, L 71-20, CP 44-101, CP 61-37 and L 62-96

(Autrey3s7). A bacterium isolated from tissues with stripes had biochemical and physio- logical properties typical of the red stripe organism but pathogenicity tests have not been carried out as yet. The disease is considered minor at this stage. Leaf scald was suspected by Baudin9 in a plot of CP 52-68 which was destroyed, but ex-

) haustive field surveys by Bendal" and A ~ t r e y ~ . ~ , ~ , ~ failed to locate leaf scald, ratoon

1 : stunting, gumming or mottled stripe diseases.

Fungal diseases

Three fungal diseases have been recorded, namely pokkah boeng (Fusarium moniliforme), red rot of the leaf sheath and mid rib (Glomerella tucumanensis), and brown spot (Cercospora longipes) (Autrey2). While the two former were present in all districts, brown spot was found only in Loukkos. The diseases are considered minor, with no impact on production.

I NON-PARASITIC DISEASES

Various non-parasitic diseases have been described in Morocco. The most severe condi- tion is undoubtedly frost damage which was considerable in 1976 and 1985 (Autrey3, Schmidt ef all8, Schmidt ef ail9). Damage by frost has been minimized by locating the sugar cane areas close to the coast, and premature harvest is recommended when it occurs (~chmidt'et aPO). Leaf fleck similar to silicon deficiency symptoms was found in cane growing on a sandy soil, and interveinal chloros~s due to iron deficiency was ob- served in the Basse Moulouya area in cane growing on alkaline soils (Autrey2). Varie- gation, a genetical defect inducing partial to total chlorosis, is more severe in spring and varieties such as L 61-67 have been found severely affected (Schmidt et aI2O). Bunch tb;

\

PATHOLOGY

was observed in various varieties but particularly in CP 65-357. It is common in certain years, in a country where owing to photoperiodicity, sugarcane does not flower.

1 QUARANTINE PROCEDURES

Morocco imports sugarcane cuttings from USA and the facilities of ClRAD in Montpelli- er, France. Fuzz is imported from the USA only. Although no pathogen has been report- ed to-date to be transmitted through fuzz, seedlings produced in the glasshouse are grown and observed in the field well outside the cane growing area for one year, be- fore transfer to the main experiment station of CTCAS in the Gharb. Both closed and open quarantine facilities are located inside the capital, Rabat, at least 80 km from the nearest commercial fields. The quarantine procedures have been described by Autrey3. Plants are grown in confinement for 18 months over one plant cane and one ratoon. Cuttings from shoots found disease-free by visual observations, diagnosis by ELlSA and immunofluorescence and electron microscopy, are given hot water treatments of 500C for 30 min and 50% for 2 hours after 24 hours. Plants from these cuttings are observed in open quarantine for 18 months during one plant cane and one ratoon. Since the establishment of these elaborate quarantine procedures in 1984, there is no evidence that any disease has been introduced through quarantine. The suspected presence of RSD in the Argentinian variety NA 6390 was not confirmed by advanced diagnostic tests (Autrey5).

NURSERY SYSTEM

As one of the main aims of CTCAS is to provide disease-free cuttings to growers, an elaborate nursery system in four stages has been established for commercial varieties with basic, primary, secondary and tertiary plots of areas of 0.2, 1.0, 10 and 100 ha respec- tively for each variety. Adequate staff with proper training ensure the smooth running of the system. Proper sanitation measures such as roguing and disinfection of knives have been adopted. To-date no seed-borne diseases have been found in the commer- cial fields issuing from the nursery plots.

I EVALUATION OF VARIETIES RELATIVE TO FOREIGN DISEASES

The greatest danger facing the Moroccan sugar industry in terms of diseases is the smut pathogen Ustilago scitaminea present in neighbouring and other African countries such as Mali, Senegal and Egypt, and which is likely to thrive in the prevailing dry condi- tions. The absence of the disease in Morocco to-date could be due to the Sahara acting as a natural barrier against air borne spores. Smut remains a constant danger as CP 44-101 and CP 65-357, the major varieties in the Gharb area are known to be susceptible to smut in Senegal and other African countries (Marion, pers comm). Commercial and promising varieties are being tested against smut in Senegal (A~ t rey~ ,~ ) , and prelimi- nary results confirmed that CP 44-101 was susceptible. Information is being gathered from various countries on the reaction of commercial and promising varieties to com- mon rust (Puccinia melanocephala) and other diseases.

I DISCUSSION AND CONCLUSION

The Moroccan sugar industry is one of the cleanest In Africa and around the world, as the recent ISSCT disease listing demonstrates (Anon'). Important diseases that thrive i.n conditions similar to those of Morocco, for example leaf scald, RSD and smut, do not occur and there is no evidence that they have been introduced. With the apparent

71 8

L.J.C. AUTREY, A. MADRANE, F.W. HESSE AND M. NADlF

eradication of SCMV-D in the field, and if the stringent quarantine procedures and elaborate nursery systems are maintained, smut and possibly SCBV are the only patho- gens of possible concern. The arrangements made for testing the reactions of varieties to smut in Senegal are therefore of considerable importance. Since two to three years would be necessary to establish the reaction of varieties to the pathogen if it is introduced, the disease would have time to contaminate any varieties which happen to be suscepti- ble. Smut evaluation work should therefore be intensified. Although SCMV appears to be under control, glasshouse tests in Morocco and field tests in Spain will be maintained so as to avoid release of varieties highly susceptible to the virus. In case such varieties have to be released to meet specific needs, care will be taken to ensure that there is no build up of SCMV.

. The presence of SCBV in one out of 17 samples taken from CP 44-101 at random in thy Gharb in worrying. Further work is being carried out to determine the incidence of the dis- ease in the field and its effect on cane and sugar yields. Assessment of varietal reac- tion, the development of a heat treatment to cure infected setts, and the elucidation of the mode of spread of the disease are considered as priorities, now that diagnostic . procedures have been established. The relationship between SCBV and the bacilliform virus found in Cuba should be es- tablished. They appear to be the same and whether coinfection with SCMV in both coun- tries is only due to chance or some other unknown factor, has to be unravelled. Although research on sugarcane diseases is recent in Morocco, it has made an interest- ing contribution to the advancement of this discipline with the identification of SCBV. Sugarcane pathologists in other countries should try to establish the status of SCBV in their cane germplasm, since it has been found in Hawaii (Lockhart and AutreyI5) and in Cuba in varieties from China, Vietnam, Laos and Mexico (Peralta, pers comm). The potential of this virus to cause yield losses, and its world distribution, needs to be deter- mined as a matter of urgency. Morocco enjoys an enviable situation in terms of incidence of diseases and their im- pact. This can only be maintained by vigilance as developments take place in the years ahead. Through the established infrastructure and the expertise and manpower train- ing provided by GTZ to CTCAS, the Moroccan sugar industry is in a privileged situation to face the challenge of staying nearly disease-free. This should allow it to fulfil the ultimate aim of its sugar production, which is the attainment of self sufficiency, a mat- ter of considerable economic importance to the country.

I REFERENCES

1. Anon (1989). Sugarcane diseases and their world distribution, pp 362-370. In C Ricaud, BT Egan, AG Gillaspie jr and CG Hughes (Eds). Diseases of sugarcane - major dis- eases. Elsevier, Amsterdam.

2. Autrey, l,JC (1983). Maladies de la canne a sucre au Maroc. Rapport a I'Office Alle- mand pour la Cooperation Technique (GTZ). 23 pp.

3. Autrey, LJC (1985). Travaux de recherche en phytopathologie et aspects connexes de la canne a sucre au Maroc. Rapport ?I I'Office Allemand pour la Cooperation Technique (GTZ). 66 pp. + annexes.

4. Autrey, LJC (1986). Poursuite des travaux en phytopathologie et aspects connexes de la canne 6 sucre au Maroc. Rapport 6 I'Office Allemand pour la Cooperation Technique (GTZ). 18 pp.

5. Autrey, LJC (1987). Mission en Espagne, au Senegal et au Maroc liee a la pathologie de la canne ~3 sucre. Rapport a I'Office Allemand pour la Cooperation Technique (GTZ). 41 pp.

PATHOLOGY

6. Autrey, LJC (1988). Poursuite des travaux sur I'evaluation des varietes de canne a

sucre cultivees au Maroc au virus de la mosaique. Rapport b I'Office Allemand pour la Cooperation Technique (GTZ). 8 pp.

7. Autrey, LJC (1988). Poursuite des travaux sur les agents pathogenes d'importance a la canne a sucre au Maroc. Rapport a I'Office Allemand pour la Cooperation Tech- nique (GTZ). 6 pp + annexes.

8. Autrey, LJC and Madrane, A (1985). Implantation en Espagne d'un essai pour evaluer la reaction des varietes de canne 6 sucre cultivees au Maroc au virus de la mosai- que. Rapport b I'Office Allemand pour la Cooperation Technique (GTZ) and I'Office Regional de Mise en Valeur Agricole du Gharb, Maroc. 17 pp.

9. Baudin, P (1976). Etat sanitaire des cultures de la canne a sucre duns la plaine du Gharb (ORMVAGARAT). Rapport de mission 22 ao0t-4 septembre 1976. 12 pp.

10. Benda, GTA (1977). Observations on sugarcane diseases in Morocco 29 May-22 June 1977. German Agency for Technical Cooperation (GTZ). 12 pp.

11 . Berthier, P (1966). les anciennes sucreries du Maroc et leurs reseaux hydrauliques. lmprimeries francaise et marocaine, Rabat.

12. Fisher, HU and Lockhart, BE (1974). Identity of a strain of sugarcane mosaic virus occurring in Morocco. Plant Dis Reptr 58: 1121-1123.

13. Grillot, G (1957). Les essais de canne a sucre au Maroc. Bulletin de la Societe des Agriculteurs du Maroc. 77: 21-29.

14. Koike, H and Gillaspie, AG (1989). Mosaic, pp 301-322. In C Ricaud, BT Egan, AG Gillaspie jr and CG Hughes (Eds). Diseases of sugarcane - major diseases. Elsevier, Amsterdam.

15. Lockhart, BEL and Autrey, LJC (1988). Occurrence in sugarcane of a bacilliform virus related serologically to banana streak virus. Plant dis Reptr 72: 230-233.

16. Madrane, A, Hesse, FW, Karmoussi, M and Odda, M (1986). Evaluation de la reac- tion des varietes de canne r i sucre cultivees au Maroc au virus de la mosaique de la canne a sucre. Office Regional de Mise en Valeur Agricole du Gharb, Maroc. 9 pp.

17. Madrane, A, Hesse, FW, and Odda, M (1986). Evaluation de la reaction des varietes de canne tr sucre cultivees au Maroc au virus de la mosaique. Office Regional de Mise en Valeur Agricole du Gharb, Mar'oc. 6 pp.

18. Schmidt, G, Hesse, FW and Trost, K (1972). Recolte de la canne a sucre en zone ge- live en automne et en hiver. Al Awania 44: 35-50.

19. Schm~dt, G, Hesse, FW, N'Ciri, A and Trost, K (1978). L'effet du fort gel en janvier 1976 sur la canne sucre duns le Gharb et la vallee du Loukkos. Al Awania 56: 1-36.

20. Schmidt, G, Hesse, FW, Moughly, E and Trost, K (1981). Lo canne b sucre au Maroc. Office Allemand pour la Cooperation Technique (GTZ). 88 pp.

1 1 L.J.C. AUTREY, A. MADRANE, F.W. HESSE ET M. NADlF

LE VIRUS BAGILLIFORME DE LA CANNE A SUCRE ET D'AUTRES I 5 % I t

MALADIES DE LA CANNE AU MAROC

L.J.C. Autrey, A. Madrane, F.W. Hesse et M. Nadif

! I Centre Technique de la Canne a Sucre Kenitra, Maroc

Mots cles: Maroc canne a sucre, maladies, champignons, mosaique, virus bacilliforme, maladies non-parasitaires, degats causbs par le gel.

Deux viroses de la canne a sucre on hte identifiees au Maroc. II s'agit d'un nouveau virus latent et mecaniquement transmissible appelB virus bacilliforme de la canne a

sucre (SCBV) dont les particules mesurent 131 X 31 nm et de la souche D du virus de la mosaique de la canne 6 sucre (SCMV-D). Des recherches ont Bt6 entreprises sur

'

I'6pidBm1ologie, les plantes hBtes, les vecteurs et la reaction varietale 6 ces maladies

Des tests d'immunodiffusion, I'ELISA et I'immunomicroscopie Blectronique ont et6 uti- lises avec succes pour leur diagnostic. Des mesures de controle ont BtB entreprises avec succes pour la mosaique qu'on ne voit plus dans les champs commerciaux tandis que I'incidence exacte et I'importance Bconomique du SCBV doivent 6tre determinees. Les trois maladies cryptogamiques identifiees, le pokkah boeng, la morve rouge et les tiches brunes sont mineures. Une incidence negligeable d'une meladie bactbrienne probablement celle des rayures rouges b Bte observee. Parmi les cinq maladies non parasitaires identifibes, la rnouci~eture des feuilles, la chlo- rose interveinale, le sommet agglomere et la panachure ne sont d'aucune importance tandis que le gel peut causer des degats considerables. Un systeme Blabore de quarantaine a BtB mis en place hors des zones oir la canne est cultivbe et un systeme de pepinibres a quatre stages fournit du materiel sain pour les plantations commerciales. Des essais pour I'evaluation de la rBsistance des varietes cultivees ou Maroc, au char- bon et au SCMV-D ont 6th implant& au S6negal et en Espagne respectivement.

VIRUS BACILIFBRME DE LA CANA DE A Z ~ ~ C A R Y OTRAS ENFERMEDADES DE LA CANA DE AZUCAR EN MARRUECOS

L.J.C. Autrey, A. Madrane, F.W. Hesse y M. Nadif

Centro Tecnico de la Cafia de Azucar, Kenitra, Marruecos

Palabras claves: Marruecos, catia de azbcar, enfermedades, hongos, SCMV, virus baciliforme, no parasiticas, datio por heladas.

RESUMEN

Se ha identificado un nuevo virus latente transmisible mecanicamente, designado co- mo virus baciliforme de la catia de azljcar (SCBV) con particulas de 131 X 31 nm y la raza D del virus del mosaico de la cana de azljcar (SCMV-D). Se iniciaron investiga- ciones sobre sus epidemiologias, rango de hospedantes, interacci6n con vectores y reac-

ci6n varietal. Para su diagnosis se han aplicado pruebas de inmunodifusi6n, ELlSA e inmuno electro microscopia. Se adoptaron satisfactoriamente medidas para el control del SCMV-D el cual no se encuentra frecuentemente en el campo mientras debe esta- blecerse la exacta distribuci6n del SCBV y su importancia econ6mica. Las tres enfermedades fungicas registradas pokkan boeng, muermo rojo y mancha ma- rron son menores. Se observ6 un nivel insignificante de lo que probablemente sea la enfermedad de la raya roja. Entre las cinco enfermedades no parasiticas identificadas leaf fleck, clorosis interner- val, escoba de bruia y variegaci6n son menores mientras que la helada pueda causar un dano considerable. Se estableci6 un elaborado sistema de cuarentena aleiado de las areas cafieras y un sistema de vivero de cuatro etapas para la provisi6n de material libre de enfermeda- des. Las pruebas de resistencia para evaluar la reacci6n varietal al carbon y al SCMV-D se ben realizado satisfactoriamente en Senegal y Esparia respectivamente.

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