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MEDEDELINGEN DER ZITTINGEN BULLETIN DES SEANCES
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KONINKLIJKE ACADEMIE
VOOR OVERZEESE WETENSCHAPPEN
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WETENSCHAPPELIJKE MEDEDELINGEN
COMMUNICATIONS SCIENTIFIQUES
on Tropical Pathogenic Fungi (Brussels, 18 February, 2004)
FUNGI AND FOOD,
NEGATIVE AND POSITIVE INTERACTIONS IN THE TROPICS
Recent development in the understanding and management of :
— Reduction of food production by plant parasitic fungi, either directly or by vectoring viruses;
— Alteration of food quality by post-harvest colonization and mycotoxin production;
— Stimulation of food production by symbiotic association;
— Food preservation and improvement by fungal biocontrol.
The Sixth Edition of the Raymond Vanbreuseghem Conference took place at the “Palais des Académies”, rue Ducale 1, 1000 Brussels, on Wednesday
18 February, 2004.
Programme
H . Ma r a it e: Introduction
M . Pe r n e e l et al. : Ecologically Sustainable Management o f the Cocoyam Root Rot Disease caused by Pythium myriotylum
A. Le g r e v e et al. : Polymyxa graminis, a Promiscuous Vector o f Viruses on Crops in Tropical Areas
F. Mu n a u t & F. Va n Ho v e : Diversity in Pathogenicity and Mycotoxin Production o f the Gibberella fujikuroi Group in the Tropics
S. De c l e r c k : The Glomerales on Bananas : Domesticated Symbionts fo r Plant Growth and Root-pathogen Biocontrol
H . Jija k l iet al. : Antagonistic Activity o f Yeast against Post-harvest Diseases o f Tropical Fruits
Meded. Zitt. K. Acad. Overzeese Wet.
Bull. Séanc. Acad. R. Sci. Outre-Mer 50 (2004-2): 99-101
Introduction
b y
Henri Ma r a it e *
Dear Colleagues, Ladies and Gentlemen,
I join Professor Yola Verhasselt, Permanent Secretary, in welcoming you to the 6th Raymond Vanbreuseghem Conference on tropical pathogenic fungi, organized by the Royal Academy of Overseas Sciences.
Before starting with the scientific programme, I would like to remind the audience, especially the younger members, of some aspects of Raymond Vanbreuseghem’s activities and the reasons why the Academy keeps alive the memory of this outstanding figure in medical mycology.
Raymond Vanbreuseghem was born in 1909 in Monceau/Sambre. In 1934 he graduated as a Medical Doctor from the “Université de Liège” and completed his training with a diploma in Tropical Medicine at the Prince Leopold Institute for Tropical Medicine in Antwerpen. He left Belgium for the Belgian Congo in
1935.
His first research interest was in bacteriology, in particular the diagnosis and treatment of leprosy. In 1946 he returned to Europe and through his interest in dermatology his work evolved towards medical mycology. During a stay with Maurice Langeron at the Medical Faculty in Paris he rounded off his expertise in mycology and, with Langeron, published the second edition of the Précis de Mycologie in 1952, the main reference textbook for many years in many myco- logical laboratories.
Raymond Vanbreuseghem was appointed Professor at the “Université Libre de Bruxelles” in 1954 and lectured in Medical and Veterinary Parasitology. In 1965, at the Prince Leopold Institute for Tropical Medicine in Antwerpen, he created a course in human and veterinary mycology. Indeed, he devoted all his passion, talent and energy to this field and made major scientific contributions published in over 350 papers.
The starting points of research topics for this medical doctor were patients suffering from unpleasant fungal infections. As an inventive scientist he devel
* M em ber o f the A cadem y ; U nité de Phytopathologie, U niversité catholique de Louvain, Croix du Sud 2/3, B-1348 Louvain-la-N euve (Belgium ).
oped ways of isolating, characterizing and describing a wide range of fungal species causing diseases in humans. Starting from the hair and the skin he tracked fungi down to the lung and deep into the heart.
In his research, Professor Vanbreuseghem also paid attention to ways of avoiding and controlling these infections. This led him to investigate possible sources of contamination with the discovery of the saprophytic phase in the soil for some of the fungi pathogenic to humans or animals. The demonstration and study of this phase was a major breakthrough in medical mycology, with spin
offs for plant pathology. Professor Vanbreuseghem popularized, for example, the concept of using simple natural substrates such as hair and soil extracts for the selective isolation and growth of dermatophytes, a concept still very pertinent in the study of plant pathogens. He also analysed the opportunistic behaviour of some fungi, a topic of prime and renewed importance in the case of AIDS-assoc
iated immune-depression, a concept also relevant to plant pathology.
His expertise was internationally recognized. He lectured worldwide and was a cofounder of the International Society of Human and Veterinary Mycology. He became a member of the Royal Academy of Colonial Sciences (now the Royal Academy of Overseas Sciences) in 1954, and demonstrated his dedication to the Academy when he was his President and as Chairman of the organizing com
mittee for the celebration of its fiftieth anniversary in 1978. Between 1980 and 1981 he served the Academy as a Supply Permanent Secretary during a very crit
ical period of this institution’s history. He laid the foundations for an improved financial situation and the implementation of new statutes.
In recognition of these outstanding contributions, the Royal Academy of Overseas Sciences awarded him the title of Honorary Permanent Secretary and in 1982 established the Raymond Vanbreuseghem Fund to keep alive, and pay tribute to, his mycological work. This Fund is used to organize public confer
ences on fungal diseases of humans, animals or plants in tropical countries. In his thanks to the Academy, Raymond Vanbreuseghem indeed expressed the wish for broadening the scope of theses conferences from his working area on human and animal fungal diseases to work on the fungal diseases of plants.
The first Conference was organized in 1987, and the third Conference was on plant fungal diseases. It was held on 3 December 1993, six days after the death of Raymond Vanbreuseghem.
After the 1996 and 2000 Conferences on human fungal pathogens, it was decided to devote the 6th Vanbreuseghem Conference again to plant-associated fungi in the tropics, making a link with human health — Professor Vanbreuseghem’s main scientific goal — through its focus on “fungi and food”.
But rather than dealing with the topic “fungi as food”, we propose to highlight some recent developments in the understanding of plant parasitic fungi having negative effects on food production, in terms of crop yield reduction through fungal parasitism (as will be highlighted by Professor Monica Höfte) or in terms of vectoring viruses, a less well-known effect (as illustrated by Dr Anne Legrève)
— 101 —
or in terms of the alteration of food quality through post-harvest colonization and mycotoxines’ production (a topic addressed by Dr Françoise Munaut).
Nevertheless, as pointed out judiciously by Professor J.-J. Symoens in his tribute to Raymond Vanbreuseghem in 1982, fungi can also have positive aspects, but not just from the culinary point of view.
Indeed, recent decades have shown the potential of some fungi to increase food production through mutualistic association with plants (as highlighted in a recent issue of Nature), or symbiotic association with plant roots (the topic of Dr Stéphane Declerck’s presentation), or to reduce post-harvest losses through biocontrol of pathogens (a topic addressed by Professor Haïssant Jijakli).
The overall aim of all theses studies is to help farmers in the tropics to limit food losses caused by fungal diseases. Indeed, beyond the mean figures of yield losses at global or regional level, the impact of an affected or spoiled staple food crop needs to be considered at the level of the farmer’s family. The speakers will therefore also show how the progress in understanding the complex fungal-plant interactions allows the development of strategies for the sustainable management of fungi with negative effects on food production through, for example, cultivar resistance, farming practices or biocontrol, as well as the development of strat
egies for the enhanced activity of those fungi with positive effects.
I wish to thank the speakers for their willingness to share their experience with us today at this 6th Raymond Vanbreuseghem Conference. I am also grate
ful to Professor Danielle Swinne, a former student of Professor Vanbreuseghem and now in charge of the Vanbreuseghem Mycotheque, for providing me with the information about this outstanding mycologist. Thanks also to the Academy staff who organized this conference.
REFERENCES
Clay, K. 2004. Fungi and th e food o f gods. — Nature, 427 (6973) : 401-402.
Hommage au Secrétaire perpétuel honoraire Raymond Vanbreuseghem / Hulde aan de Erevaste Secretaris Raymond Vanbreuseghem 1983. Bruxelles, Académie Royale des Sciences d’Outre-Mer / Koninklijke Academie voor Overzeese Wetenschappen, 72 pp.
Sw in n e, D. 1994. Obituary R. Vanbreuseghem 1909-1993. — M ycopathologia, 128 : 65.
50 (2 0 0 4 -2 ): 103-113.
Ecologically Sustainable Management of the
Cocoyam Root Rot Disease caused by Pythium myriotylum *
by
Maaike Pe r n e e l**, Amayana Ad io b o **, Caroline Fl o r e n **, Katrien De Ma e y e r**, Annelies Ve r c a u t e r e n **,
Francisco Sa b o r io *** & Monica Ho f t e **
Ke y w o r d s. — Xanthosom a sagittifolium ; Genetic Characterization ; AFLP ; Biological Control ; Compost.
Su m m a ry. — Cocoyam (Xanthosoma sagittifolium), an important staple food for mil
lions o f people in the tropics suffers from the root rot disease caused by Pythium m yrio
tylum. In Cameroon severe attacks have been observed, but no adequate control measure has been found so far. In this report preliminary results on the characterization o f the cocoyam root rot pathogen and a biological control strategy are presented. AFLP analysis and pathogenicity tests revealed that P. myriotylum isolates from cocoyam are quite dis
tinct from P. myriotylum isolates from other hosts and have probably developed a specific host adaptation to cocoyam. Two phenazine-producing Pseudomonas strains, isolated from the rhizosphere o f healthy cocoyams, were very effective against P. myriotylum in vitro. A survey about cultural practices in Cameroon revealed that minimum tilled lands had the lowest disease incidence. Greenhouse experiments demonstrated that compost, prepared from locally available organic matter, inhibited infection o f P. myriotylum on cocoyam.
Tr efw o o r d en. — Xanthosoma sagittifolium ; Genetische karakterisatie ; AFLP ; Biologische controle ; Compost.
Samenvatting. — Ecologische duurzame bestrijding van de wortelrotziekte op cocoyam veroorzaakt door Pythium myriotylum. — Cocoyam (Xanthosoma sagittifo
lium), een belangrijk voedingsgewas voor miljoenen mensen in de tropen, heeft af te reke
nen met wortelrot veroorzaakt door Pythium myriotylum. In Kameroen werden er grote verliezen geobserveerd maar tot op heden zijn er nog geen doeltreffende bestrijdings- maatregelingen gevonden. In dit rapport worden preliminaire resultaten van de karakteri
satie van de wortelrotpathogeen op cocoyam en een biologische bestrijdingstrategie voorgesteld. AFLP-analyse en pathogeniciteitstesten toonden aan dat P. myriotylum iso- laten van cocoyam zeer verschillend zijn van P. myriotylum isolaten van andere waard- planten en waarschijnlijk een specifieke waardplantadaptatie voor cocoyam ontwikkeld
* Paper read at the Sixth Raym ond Vanbreuseghem Conference held on 18 February 2004. Text received on 22 M arch 2004.
** Laboratory o f Phytopathology, D epartm ent o f C rop Protection, Faculty o f A gricultural and A pplied B iological Sciences, G hent University, C oupure Links 653, B-9000 G hent (Belgium ).
*** U niversidad de C osta Rica, San José (C osta Rica).
— 104 —
hebben. Twee fenazine-producerende Pseudomonas stammen, geïsoleerd uit de rhizosfeer van gezonde cocoyams, bleken zeer effectief te zijn tegen P. myriotylum in vitro.
Veldonderzoek in Kameroen toonde aan dat gronden waarop minimale grondbewerking is uitgevoerd, de laagste infectiedruk vertoonden. Plantexperimenten toonden aan dat de inwerking van compost, bereid met lokaal beschikbaar organisch afval, de infectie van P. myriotylum op cocoyam kon verhinderen.
Mo t s-c l e s. — Xanthosoma sagittifolium ; Caractérisation génétique ; AFLP ; Controle biologique ; Compost.
Re su m e. — Gestion écologiquement durable de la pourriture racinaire du macabo causée p a r Pythium myriotylum. — Le macabo (Xanthosoma sagittifolium) est une plante vivrière de base pour des millions de populations des tropiques. Mais il est l’objet d’atta
ques de la pourriture racinaire causée par Pythium myriotylum. Le Cameroun fait partie des pays où les attaques dues à cette maladie sont les plus sévères et où il n’existe tou
jours pas de méthodes effectives de contrôle. Dans ce rapport, les résultats préliminaires obtenus sur la caractérisation et la stratégie de contrôle biologique de ce pathogène sont présentés. L’analyse AFLP et des tests de pathogénicité révèlent des différences entre les isolats de P. myriotylum obtenus du macabo et ceux provenant d’autre plantes hôtes, sug
gérant ainsi l’hypothèse d’une adaptation spécifique du pathogène au macabo. Par ailleurs, deux souches de Pseudomonas productrices d’antibiotiques (Phenazines) isolées de la rhizosphère des plants sains du macabo, se sont avérées efficaces contre le Pythium in vitro. Une enquête sur les pratiques culturales au Cameroun a montré que la faible inci
dence de la maladie était fortement associée aux sols faiblement labourés. D es expéri
mentations menées en serre ont montré que le compost préparé sur base du matériel organique local empêche l’infection du Pythium sur le macabo.
Introduction
Cocoyam (Xanthosoma sagittifolium [L ] Schott), a member of the Araceae family, is a staple food for more than 400 million people living in the tropics and subtropics ( Ly o n g a 1980, On k o p is eet al. 1992). Cocoyam is widely cultivated in West-African countries, Central America and the Caribbean, Oceania and Asia.
It is mainly cultivated for its cormels which provide carbohydrate in human diet, and also contains significant amounts of proteins, fat and essential vitamins
( Co b l e y & St e e l e 1986). In fact, cocoyam has a higher content of protein and sulphur-bearing amino acids than many other tropical root crops like cassava
( Kay 1987) and the food-energy yield of cocoyams per unit land area is very high
(Pa r k i n s o n 1984). In Cameroon, cocoyam is the second most important root crop after cassava ( Ly o n g a 1980). The cormels are processed in various traditional dishes such as fufu and the leaves are prepared as green vegetables.
Despite the importance of cocoyam as a staple food, the production of coco
yam is impaired by poor management, ignorance, lack of scientific research and diseases. Especially the cocoyam root rot disease (CRRD), caused by the soil- borne pathogen Pythium myriotylum, can cause yield reductions up to 90 %
( N z i e t c h u e n g 1983). In Cameroon, the production dropped from 1.8 to 0.8 mil
lion tons per year over a time period of ten years due to the root rot disease. In addition, infested fields can no longer be exploited, which results in reclamation of virgin soils and rapid deforestation.
Pythium myriotylum is a soil-borne pathogen with a broad host range.
P. myriotylum causes pre-emerge damping-off of seedlings including tobacco, black locust, rice, bean, groundnut and watermelon ; seedling rot of lucerne, pawpaw and tomato ; fruit rot of watermelon, cucumber and eggplant ; and root rot on ginger, oil palm, strawberry and cocoyam ( Ha l l 1993, Nz ie t c h u e n g 1983,
Wa n g et al. 2003). The pathogen is endemic in the tropics (Africa, Asia, Australia and Latin America) and the disease incidence is the highest in virgin soil containing abundant decomposing organic matter ( Ha l l 1993). The broad eco-geographic range and host range imply that Pythium myriotylum is genet
ically diverse.
A first indication of intraspecific variability within the species Pythium myriotylum was observed by Ta m b o n g et al. (1999). Unlike the other Pythium myriotylum strains, Pythium myriotylum isolates from cocoyam were unable to grow at 37 °C. Isozyme patterns could differentiate the cocoyam isolates from the other Pythium myriotylum strains. Finally, cocoyam isolates were only patho
genic to cocoyam and tobacco, while other Pythium myriotylum strains could infect tomato, bean, cowpea and tobacco. These observations suggest there might be an intraspecific variability among cocoyam isolates and Pythium myrio
tylum isolates derived from other host plants.
There is an urgent need for an adequately sustainable strategy to reduce the impact o f the root rot pathogen on cocoyam . Fungicides have not been fully effective against CRRD (Nzietchueng 1983). Breeding for resistant cocoyam s is difficult because o f ploidy incompatibility between resistant and susceptible cocoyam varieties. B iological control based on the incorporation o f organic mat
ter, with or without enrichment with antagonistic micro-organisms, may offer an ecological sustainable solution for the cocoyam root rot disease. It is known for many years that soil-borne pathogens such as Pythium sp. and Phytophthora sp.
can be suppressed by the incorporation o f organic matter (com post, green manures, litter) in soil. The disease suppressive capacity is directly linked to the total microbial activity o f that organic matter (Hoitinck & Boehm 1999).
Tambong & Hofte (2001) demonstrated that Pseudomonas aeruginosa PNA1 significantly suppressed the root rot pathogen Pythium myriotylum in the pres
ence o f cocoyam plantlets. The antagonism o f PNA1 was assigned to the prod
uction o f phenazine antibiotics which are very active against Pythium sp. and Fusarium (Anjaiah et al. 1998). However, clinical isolates o f Pseudomonas aeruginosa are opportunistic pathogens o f human beings, so the application o f this strain in the biological control o f cocoyam root rot disease is irresponsible and thus unjustified. Other non-pathogenic phenazine-producing pseudomonads which are as effective as PNA1 could be applied as biological agents.
— 106 —
A five-year research project has been established since 2001 between the lab
oratory of phytopathology at Ghent University and the “Institut de Recherche Agricole et de Développement (IRAD)”, Ekona in Cameroon. This project, sponsored by the VLIR, is entitled : “Ecologically sustainable management of the root rot disease caused by Pythium myriotylum". The main aims of the proj
ect are (1) further characterization of Pythium myriotylum isolates of cocoyam ; (2) influence of cultural practices on the occurrence of CRRD ; (3) sustainable control of CRRD by organic matter incorporation combined with effective microbial antagonists. In this paper, we will present some of the results that have already been obtained.
Results and Discussion
C o l l e c t i o n o f Py t h iu mm y r io t y l u m S t r a i n s
For the characterization of the root rot pathogen on cocoyam, a large Pythium myriotylum collection was founded in the laboratory of phytopathology at Ghent University. Pythium myriotylum strains from cocoyam were isolated single- handed or on request in Cameroon, Costa Rica and Papua New Guinea accord
ing to Pe r n e e l ( 2 0 0 2 ) . Three other Pythium strains were included in the charac
terization study as an “outgroup taxa”. Table 1 provides information about the Pythium isolates used in this study.
G e n e t i c C h a r a c t e r i z a t i o n o f Py t h iu mm y r io t y l u m u s i n g A F L P A n a l y s i s
AFLP analysis was performed on all Pythium isolates (tab. 1) as described by
Sa v e y n (2003). Five primer combinations were tested. The DNA-fingerprints obtained were scored for the presence (1) or absence (0) of electrophoretic AFLP bands for each isolate. The resulting data matrix was analysed using NTSYS-pc (version 2.10L ; Ro h l f 2000) and Treecon (Version 1.3b ; Va n d e Pe e r & De
Wa c h t e r 1994).
Figure 1 shows the dendrogram obtained with Jaccard’s coefficient and UPGMA clustering method in NTSYS-pc and Treecon. Isolates from cocoyam (cluster 1) formed a tight, homogenous group (< 15 % dissimilarity), which could be easily distinguished from the P. myriotylum isolates from culture col
lections (cluster 2). Both groups could on their turn easily be distinguished from the outgroup (cluster 3) at a genetic dissimilarity level of 0.77. Two cocoyam isolates, CMR22 and CMR25, cluster together with Pythium myriotylum isolates from culture collections. Interestingly, CMR22 and CMR25 were the only cocoyam isolates which were able to grow at 37 °C as observed for Pythium myriotylum strains from other host plants ( Ta m b o n g et al. 1999). The outgroup also contains two cocoyam isolates (CMR17, C l03-04) which are probably not Pythium myriotylum strains. These findings prove that AFLP is a powerful tech-
T able 1
B rief overview o f Pythium strains involved in the characterization study
N am e H ost O rig in R eference
P ythium splendens m onstera roots Belgium Buysenset al. 1996
Pythium m yt 14 soil Cam eroon Tambonget al. 1999
Pythium NS2 soil Cameroon Tambonget al. 1999
P. m yriotylum IM I 061432R pineapple Australia International Mycological Institute, UK P. m yriotylum IM I 248096 sorghum A ustralia International Mycological Institute, UK P. m yriotylum tobacco tobacco USA U niversity o f Tennessee, USA P. m yriotylum P-150 gypsophila Israel The Volcani Center, Israel P. m yriotylum P-243 groundnut Israel The Volcani Center, Israel P. m yriotylum P-249 gypsophila Israel The Volcani Center, Israel P. m yriotylum P-256 anigozanthos Israel The Volcani Center, Israel P. m yriotylum M U CL groundnut Israel M ycothèque de l’U niversité
P m l6 1 6 6 Catholique de Louvain
P. m yriotylum CBS 315.33 stem rot tobacco
Netherlands Centraalbureau voor Schim m elcultures
P. m yriotylum CBS 695.79 bean M alaysia C entraalbureau voor Schim m elcultures P. m yriotylum crum ley groundnut USA A& M Texas University, USA P. m yriotylum CRPm cocoyam Cam eroon Tambonget al. 1999 P. m yriotylum Bokwai cocoyam Cam eroon Tambonget al. 1999 28 Pythium m yriotylum cocoyam Cam eroon Perneel 2002, Saveyn 2003
isolates(CM R 1-CM R26)
11 Pythium m yriotylum cocoyam Costa Rica This study
isolates (CR1-CR11)
Pythium m yriotylum C l 03-04 cocoyam Cook Islands Jacqui W right, Cook Islands
nique to characterize Pythium myriotylum cocoyam isolates since AFLP gener
ates many polymorphic markers in a short time period. This results in a very reliable dendrogram (cocoyam cluster is 100 % reliable according to bootstrap analysis).
P a t h o g e n i c i t y o f Py t h iu m m y r io t y l u m o n C o c o y a m
The pathogenicity of the Pythium myriotylum strains was tested on tissue- derived cocoyam plantlets in a potting mixture (Black Peat, Klasmann substrate 3:1) which was conducive to cocoyam root rot. Four cocoyam isolates from Cameroon (CMR1, CMR15, CMR17, CMR25), two cocoyam isolates from Costa Rica (CR4, CR9), one cocoyam isolate from Cook Islands (C l03-04) and five Pythium myriotylum strains from other host crops were included. The plant
ing medium was infested with 1,250 propagules/g soil, except for the control.
For each treatment five cocoyam plantlets were used. After seven days the leaves were scored for disease severity using a rating scale from 1 = no visible symp
toms to 5 = death of the leaf. Collected data were analysed with the Kruskall Wallis and Mann U Whitney test using SPSS (version 11.0) statistical software.
Figure 2 shows the percentages of diseased cocoyam leaves in each class after inoculation with different Pythium myriotylum isolates. Isolates from
— 108 —
Fig. 1. — D endrogram show ing the genetic relationship am ong Pythium m yriotylum strains based on A FLP-analysis, using the Jaccard’s coefficient and U PG M A clustering m ethod. A bootstrap analysis was perform ed to test the reliability o f the clusters. O nly bootstrap values o f 50 % and higher are indicated. The scale on top shows the degree o f dissim ilarity.
cocoyam irrespectively of their origin, were highly pathogenic to cocoyam plantlets. However, Pythium myriotylum strains from other host crops showed no pathogenicity to cocoyam plants. Three cocoyam isolates (CMR17, CMR25, C l03-04) from Cameroon and Cook Islands, which did not cluster with the cocoyam isolates in figure 1, were not pathogenic to cocoyam either. Apparently the significant differences observed on DNA-level for these three cocoyam strains were also reflected in their pathogenicity. This finding confirms that these strains are non-pathogenic Pythium species isolated from the rhizosphere of cocoyam plants. The pathogenicity test clearly demonstrates that only isolates initially isolated from cocoyam, are pathogenic to cocoyam while other Pythium myriotylum strains were not able to infect cocoyam. In addition, Ta m b o n g et al.
(1999) observed an extremely narrow host range and a different growth optimum for cocoyam isolates. The different characteristics of the cocoyam isolates sug
gest a specific host adaptation to cocoyam.
Pathogenicity test
Fig. 2. — Pathogenicity o f tw elve different Pythium m yriotylum strains on tissue culture-derived plantlets. Cocoyam plantlets w ere inoculated at a dosis o f 1,250 P. m yriotylum propagules/g o f soil and w ere evaluated after seven days. T he leaves w ere scored using a rating scale from 1 = no visible sym ptom s to 5 = death o f the leave and statistically analysed using SPSS. Bars w ith different letters are significantly different at 5 % level using M ann U W hitney Test.
Ef f e c t o f Cu l t u r a l Pr a c t ic e s o n t h e Oc c u r r e n c e o f t h e Co c o y a m Ro o t Ro t Dis e a s e
Over a ten-month time period, about sixty-five cocoyam farms in three eco
logical zones in Cameroon were selected for monitoring the CRRD development in relation to cultural practices. Data were collected on soil types, land prepara
tion techniques, cropping system and the amount of crop residues generated and incorporated in the soil. A preliminary survey demonstrated that CRRD occurred in all types of soil irrespectively of the topography (upland, lowland, bottomland, swampland). The traditional “slash-and-bum” technique and deep ploughing favoured the development of CRRD. On the contrary, minimum tilled forest- derived lands without burning were suppressive to CRRD, probably due to the higher amounts of fresh organic matter. This survey shows that some cultural practices seriously influenced the CRRD severity and should thus be avoided.
Minimum tillage as land preparation method however should be encouraged.
Sc r e e n in gf o r Fl u o r e s c e n t An t a g o n is t ic Rh iz o b a c t e r ia
Fluorescent pseudomonads were isolated from the rhizosphere of healthy cocoyams, appearing in infested fields as described in Pe r n e e l ( 2 0 0 2 ) . The antagonism of the fluorescent strains was tested in dual culture with Pythium
— 1 1 0 —
myriotylum according to Ta m b o n g & Ho f t e (2001). Two strains (CMR5c and CMR12a) significantly suppressed the growth of P. myriotylum and Fusarium oxysporum in vitro. The metabolites involved in suppression of Pythium and Fusarium are not yet elucidated, but preliminary results from Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) indicated the production of phenazines for both strains. In addition, HPLC also revealed the production of pyrrolnitrin and pyoluteorin for CMR5c.
Pr o d u c t io n o f Dif f e r e n t Co m p o s t Ty p e s
In Cameroon, organic materials such as domestic and industrial waste were applied to produce compost (tab. 2). Beds of different feedstock were alternate
ly laid out to form compost piles. The starter was either manure or topsoil.
During composting, temperature and moisture were daily checked. Moisture lev
els varied between 40-70 %, while the temperature was maintained around 70 °C. Compost piles were manually turned when necessary and were passively aerated. The compost was considered mature when the temperature stabilized at 40-45 °C.
T a b ic 2
C om position o f the different com post types prepared in Cam eroon C o m p o st
1 2 3 5 6 7 9
C o m p o sitio n
Green grasses X X X X
B anana stems X X
Sugarcane waste X X X X X
Tree branches-leaves X X
M anure/fow l waste X X X X X X X
Cacao pods X
Coffee hulls X X
Oil palm waste X X
Tree barks X X
Su p p r e s s iv e n e s s o f Co m p o s t t o Ro o t Ro t Pa t h o g e n
The suppressiveness of seven different composts was tested in the presence of tissue-derived cocoyam plantlets in black peat, a very conducive substrate to cocoyam root rot. Compost was mixed with black peat in a ratio of 1:4. The planting medium was infested with 1,250 propagules/g soil of Pythium myrio-
tylum CMR1, except for the healthy control. For each treatment five cocoyam plantlets were used. After seven days the leaves were scored for disease severity using a rating scale from 1 = no visible symptoms to 5 = death of the leaf.
Collected data were analysed with the Kruskall Wallis and Mann U Whitney test using SPSS (version 11.0) statistical software.
Figure 3 shows the effect of seven different types of composts on the root rot pathogen Pythium myriotylum CMR1. Composts 2 and 5 were not significantly different from the diseased control (BP). Compost 6 showed an intermediate sup
pression while composts 1, 3, 7 and 9 were very suppressive to Pythium myrio- tylum. In fact, the treatment with compost 7 did not differ significantly from the healthy control. This test clearly indicates that incorporation of organic matter can reduce the impact of the root rot pathogen on cocoyam and in some cases even eradicate the effect of the pathogen. The mechanism of suppression cannot yet be explained, although there are some indications that the suppressiveness is directly linked to the microbial activity in the compost, since sterile compost lost its suppressiveness.
S u p p ressiven ess of co m p o st
fg cd e f be
(/)CD
>TO
<D n0)
ED
C
100%
80%
60%
40%
20% 0%
■ Class 5
■ Class 4 sa Class 3
□ Class 2
□ Class 1
Fig. 3. — Effect o f seven different types o f com post and K lasm ann substrate on the root rot pathogen Pythium m yriotylum on cocoyam . Com post w as m ixed w ith black peat (BP) in ratio 1 :4, planting m edium was infected w ith 1,250 P. m yriotylum propagules/g soil. T he leaves w ere scored using a rating scale from 1 = no visible sym ptom s to 5 = death o f the leave and statistically analysed, using SPSS. Bars with different letters are significantly different at 5 % level using M ann U W hitney Test.
- 1 1 2 -
Conclusions
AFLP analysis and pathogenicity tests have revealed that P. myriotylum iso
lates from cocoyam are quite distinct from P. myriotylum isolates from other hosts and have probably developed a specific host adaptation to cocoyam. Two Pseudomonas strains that produce phenazine antibiotics, show great promise as biocontrol agents against P. myriotylum. Finally, disease suppressive composts have been identified and can be incorporated in the soil to reduce the impact of the root rot pathogen on cocoyam.
ACKNOWLEDGEMENTS
This work was supported by the “Vlaamse Interuniversitaire Raad (VLIR)” and
“Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWT)”. The authors wish to express their sincere thanks to Bonnie Ownley from the University o f Tennessee (USA), Terry Wheeler from A&M Texas University (USA), Ben Yephet from the Volcani Center (Israel), Francisco Saborio from the University o f Costa Rica (Costa Rica) and Jacqui Wright for the supply o f the Pythium myriotylum strains.
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Polymyxa graminis,
a Promiscuous Vector of Viruses on Crops in Tropical Areas *
by
Anne Le g r e v e **, Philippe De l f o s s e ***, Thomas Va n k e e r b e r g h e n **,
Viviane Va n He s e ** & Henri Ma r a it e **
Key w o r d s. — Plasmodiophorid ; Form Species and Control Strategies.
Su m m a ry. — Polymyxa graminis is an obligate root endoparasite mainly o f mono- cotyledonous plants. It does not cause direct damage but is the vector of up to fifteen plant pathogenic viruses. Three o f them occur in the tropics : Indian peanut du m p virus in the Indian subcontinent, Peanut clump virus in Africa and Rice stripe necrosis virus in Africa and South and Central America. Development o f PCR methods for detecting and quan
tifying P. graminis has confirmed the role o f this parasite in the transmission o f viruses to groundnut, and allowed a determination o f the plant species playing a key role in the carry-over and spread o f the disease. These studies have revealed an unsuspected promis
cuous behaviour for P. graminis ; by multiplying extensively in cereals but also by infect
ing (with or without developing further) a wide range o f species including even fortuitous hosts such as groundnut and other dicotyledonous crops, this protist acquires, carries, prot
ects in its resting spores over many years and transmits viruses depending on the vegeta
tion pattern. The outcome is that several crop or weed species, not normally affected by the viral infection, actually influence the epidemiology o f the disease by participating in the build-up or depletion o f viruliferous inoculum. Strategies such as crop rotation, varie
tal selection or trap cropping were therefore developed to manage P. graminis. Never
theless, these strategies need to be adjusted to deal with the prevailing form species of P. graminis that are characterized by specific ecological and molecular features, and by differences in their host ranges.
Mots-c l e s. — Plasmodiophoromycète ; Formes Spéciales et Stratégies de Contrôle.
Re s u m e. — Polymyxa graminis, un vecteur opportuniste de virus aux cultures en régions tropicales. — Polymyxa graminis est un parasite obligatoire intracellulaire des racines de plantes monocotylédones principalement. Il ne provoque pas de dégâts aux plantes infectées mais il est le vecteur d’une quinzaine de virus phytopathogènes, dont trois en régions tropicales : Y Indian peanut clump virus dans le sous-continent indien, le Peanut clump virus en Afrique et le Rice stripe necrosis virus en Afrique et en Amérique
* Paper read at the Sixth Raym ond Vanbreuseghem Conference held on 18 February 2004. Text received on 22 April 2004.
** Unité de Phytopathologie, U niversité C atholique de Louvain, Croix du Sud 2/3, B-1348 Louvain-la-N euve (Belgium ).
*** International C rops R esearch Institute for the Sem i-A rid Tropics (ICRISAT), BP 12404, Niam ey (Niger).
centrale et latine. Les développements récents de méthodes moléculaires de détection et de quantification de P. graminis par PCR ont permis de confirmer le rôle de ce parasite dans la transmission des virus à l’arachide et de déterminer les espèces végétales qui jouent un rôle important dans le maintien et la dissémination de la maladie. Les recher
ches ont ainsi dévoilé le comportement opportuniste insoupçonné de P. graminis ; en se multipliant abondamment sur céréales mais aussi en infectant (poursuivant ou non son développement) un large spectre d’espèces, comprenant même des hôtes fortuits comme l’arachide ou d ’autres espèces dicotylédones, ce protiste acquiert le virus, le porte, le préserve au sein de ses spores de survie pendant de nombreuses années et le transmet selon le type de végétation. Plusieurs espèces, même non affectées par l’infection virale, jouent donc un rôle prépondérant dans 1’épidémiologie de la maladie en participant à la construction ou à l’épuisement de l’inoculum virulifère. Des stratégies de contrôle comme la rotation des cultures ou la sélection variétale ont ainsi été imaginées pour combattre le comportement insidieux de P. graminis. Elles doivent néanmoins être adaptées en fonc
tion de la forme spéciale de P. graminis en cause, chacune des formes ayant des carac
téristiques écologiques et moléculaires spécifiques ainsi qu’un spectre d’hôtes propre.
Tr efw o o r d en. — Plasmodiophoromycetes ; Formae spéciales en Controlestrategieën.
Samenvatting. — Polymyxa graminis, een opportunistische virusvector op gewassen in tropische streken. — Polymyxa graminis is een obligaat endoparasiet van de wortels, voornamelijk van monocotyle planten. Dit organisme veroorzaakt geen directe schade aan de planten, doch is vector van een vijftiental plantpathogene virussen. Drie hiervan wor
den teruggevonden in tropische gebieden : het Indian peanut clump virus op het Indische subcontinent, het Peanut clump virus in Afrika, en het Rice stripe necrosis virus in Afrika en Zuid- en Centraal-Amerika. De recente ontwikkeling van moleculaire detectie- en kwantificatiemethoden maakt het mogelijk om de rol van P. graminis te bevestigen bij de transmissie naar de aardnoot, en de plantensoorten te bepalen die een rol spelen bij het in stand houden en de verspreiding van de ziekte. Het onderzoek heeft het opportunistische gedrag van P. graminis aangetoond, dat zich overvloedig vermenigvuldigt in graange
wassen. Daarnaast werd ook aangetoond dat een breed waardplantenspectrum aangetast kan worden, al dan niet met een volledige ontwikkeling. Er worden zelfs onverwachte planten als gastheer aangetroffen, zoals aardnoot en andere dicotyle gewassen. Deze pro- tist kan het virus verwerven, het gedurende jaren in stand houden via zijn duursporen en het overdragen in functie van het type plant. Als gevolg hiervan kunnen meerdere cultuur
planten o f onkruiden, zelfs indien ze zelf niet aangetast zijn, een belangrijke rol spelen in de epidemiologie van het virus door het in stand houden en het verspreiden van het viraal inoculum. Controlestrategieën werden ontwikkeld om het geniepig gedrag van P. grami
nis te omzeilen, zoals selectie van cultivars, vruchtwisseling van aardnoot met dicotyle gewassen. Deze strategie moet echter aangepast worden in functie van de aanwezige form a spéciales van P. graminis met specifiek ecologische en moleculaire karakte
ristieken en waardplantenspectrum.
Introduction
Polymyxa graminis Ledingham, an obligate root endoparasite of Gramineae, is well known as the vector of viruses causing economically important diseases
- 1 1 7-
in cereals. This vector is also associated with the transmission of pecluviruses causing peanut clump disease, although P. graminis is very rarely detected in the roots of groundnut (Arachis hypogaea L.). Research was conducted to better understand the epidemiology of peanut clump disease in India and West Africa and to improve our knowledge of the ecological and molecular characteristics of the vector in order to develop control strategies. The characterization studies enable us to show that the transmission model of pecluviruses by P. graminis is much more complex than is usually described for the transmission of viruses to cereals by this vector. In this paper, following a general description of P. gramin
is, we describe the specific features of the P. g rammis-pecluvirus interaction, the adequate control strategies that could be developed on the basis of these new elements, and how the molecular characterization work helped us in these tasks.
Taxonomical Status, Distribution and Life Cycle of P. graminis
P. graminis belongs to the order Plasmodiophorida (plasmodiophorids), a group of obligate intracellular parasites sharing common remarkable features, e.g., mitotic cruciform nuclear division and mode of infection of host cells by zoospore ( Br a s e l t o n 1995). The proper taxonomic placement of this order has been in question for many years. Initially considered as a member of the Fungi, this order was later placed in the Protozoa or the Protista. Recent phylogenetic analyses suggested that plasmodiophorids are related to the protist phylum Cercozoa ( Bu l m a net al. 2001, Ar c h ib a l d & Ke e l in g2004). Three genera in this group — Plasmodiophora, Spongospora and Polymyxa — are obligate root endoparasites and economically important because they are plant pathogens and/or vectors of plant pathogenic viruses.
P. graminis occurs worldwide. In temperate areas, it is responsible for the transmission of bymoviruses on wheat, rye, oats and sorghum, and furoviruses on barley, wheat, rice and oats. It is also associated with the transmission of Rice stripe necrosis virus (RSNV) in Africa and in Central and South America, and of two pecluviruses, the Indian peanut clump virus (IPCV) in the Indian subcon
tinent and the Peanut clump virus (PCV) in Africa.
The diseases caused by these viruses share two main characteristics related to the life cycle of P. graminis : a patchy distribution and the recurrence in the same places in the field on successive susceptible crops. In the absence of a host plant, this vector survives in the soil for several years in the form of clusters of thick-walled resting spores (= sporosori) (fig. 1). These are produced in the root cells of a host plant and are released into soil when the roots decay. They con
tribute to the inoculum potential of P. graminis and of the virus when sporosori are carrying the virus. In the presence of plant roots, resting spores can germinate and produce biflagellate zoospores, which swim in the free water to reach an epidermal root cell or a root hair. The process of root penetration is accomplished
