This art icle was downloaded by: [ P Ray] On: 16 Novem ber 2012, At : 19: 00 Publisher: Taylor & Francis I nform a Lt d Regist ered in England and Wales Regist ered Num ber: 1072954 Regist ered office: Mort im er House, 37- 41 Mort im er St reet , London W1T 3JH, UK African Journal of Aquatic Science Publicat ion det ails, including inst ruct ions f or aut hors and subscript ion inf ormat ion: ht t p: / / www. t andf online. com/ loi/ t aas20 Fungi associated with Eichhornia crassipes in South Africa and their pathogenicity under controlled conditions P Ray a & MP Hill a a Depart ment of Zoology and Ent omology, Rhodes Universit y, PO Box 94, Grahamst own, 6140, Sout h Af rica To cite this article: P Ray & MP Hill (2012): Fungi associat ed wit h Eichhornia crassipes in Sout h Af rica and t heir pat hogenicit y under cont rolled condit ions, Af rican Journal of Aquat ic Science, 37: 3, 323-331 To link to this article: ht t p: / / dx. doi. org/ 10. 2989/ 16085914. 2012. 712912 PLEASE SCROLL DOWN FOR ARTI CLE Full t erm s and condit ions of use: ht t p: / / www.t andfonline.com / page/ t erm s- and- condit ions This art icle m ay be used for research, t eaching, and privat e st udy purposes. 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The publisher shall not be liable for any loss, act ions, claim s, proceedings, dem and, or cost s or dam ages what soever or howsoever caused arising direct ly or indirect ly in connect ion wit h or arising out of t he use of t his m at erial. African Journal of Aquatic Science 2012, 37(3): 323–331 Printed in South Africa — All rights reserved Copyright © NISC (Pty) Ltd AFRICAN JOURNAL OF AQUATIC SCIENCE ISSN 1608-5914 EISSN 1727-9364 http://dx.doi.org/10.2989/16085914.2012.712912 Fungi associated with Eichhornia crassipes in South Africa and their pathogenicity under controlled conditions P Ray* and MP Hill Downloaded by [P Ray] at 19:00 16 November 2012 Department of Zoology and Entomology, Rhodes University, PO Box 94, Grahamstown 6140, South Africa * Corresponding author, e-mail: puja.ray@gmail.com Eichhornia crassipes Mart. Solms-Laubach (Pontederiaceae), water hyacinth, continues to be the world’s worst aquatic weed. In South Africa, considerable research has been conducted on biological control agents associated with water hyacinth, with the release of six arthropods and one fungus, but little is known about the occurrence and impacts of native phytopathogenic fungi. Nation-wide surveys were conducted in 2010 and 2011 on various aquatic bodies of South Africa to identify the fungal pathogens associated with water hyacinth. Diseased plant parts were collected and fungi were isolated and identified. Some 250 isolates belonging to more than 25 genera were collected. Some of these represent new host records, as well as undescribed taxa. Isolates of Acremonium zonatum (Sawada) Gams, Alternaria eichhorniae Nag Raj and Ponnappa, Bipolaris hawaiiensis (M.B. Ellis) Uchida and Aragaki, Fusarium Link, Myrothecium roridum Tode ex Fr. and Ulocladium sp., showed the highest pathogenicity and have the potential to be useful in complementing the ongoing biocontrol programme on water hyacinth in South Africa. Keywords: biological control, mycoherbicides, plant pathogens, water hyacinth Introduction The aggressive aquatic macrophyte water hyacinth, Eichhornia crassipes Mart. Solms-Laubach (Pontederiaceae), is a free-floating aquatic plant of South American origin. Currently, the plant is widely distributed throughout the tropics and subtropics and ranks as one of the most notorious aquatic weeds worldwide (Holm et al. 1977, Gopal 1987). It forms dense impenetrable mats of vegetation in freshwater bodies including rivers, lakes and dams, and irrigation and flood canals, where it impedes water flow, irrigation and navigation, and increases eutrophication, biodiversity loss and the mosquito vectors of malaria, encephalitis and filariasis (Terry 1996, Center et al. 2002). The problems are most severe in developing countries, where human activities and livelihoods are closely linked to the freshwater bodies. In South Africa it was first recorded in the early 1900s. Since then the weed has become invasive throughout the country, mainly as a result of human activities (Jacot Guillarmod 1979). Conventional methods of control rely on mechanical/ manual removal and herbicides and are inadequate and expensive measures to apply on a large scale. Therefore, biological control has been considered to be a major, long-term option for water hyacinth. Among the various biological control agents of water hyacinth, several phytopathogenic fungi have been successful (Charudattan 2001). There have been various studies on the isolation, identification and pathogenicity of fungi associated with the weed in its native range, as well as in several water hyacinth infested areas of the world (Freeman et al. 1981, Hettiarachchi et al. 1983, Jimenez and Charudattan 1998, Naseema and Balakrishnan 2001, Daddy et al. 2003, Praveena and Naseema 2004, Okunowo et al. 2008, Ray et al. 2008a) but not in South Africa. Some of the widely reported fungi infecting water hyacinth include Fusarium equiseti (Corda) Sacc., Corticium sesakii (Shirai) Matsumoto, Cephalosporium eichhorniae Padwick, Rhizoctonia solani Kuhn (Nag Raj and Ponnappa 1970, Freeman and Zettler 1971), Uredo eichhorniae Gonz Frag and Cif. (Charudattan and Conway 1975, Charudattan et al. 1976), Alternaria eichhorniae Nag Raj and Ponnappa (Nag Raj and Ponnappa 1970, Shabana et al. 1995a, 1995b), Bipolaris stenospila (Drechsler) Shoemaker (Charudattan et al. 1976), Acremonium zonatum (Sawada) Gams (Rintz 1973, Martyn and Freeman 1978), Cercospora piaropi Tharp. (= C. rodmanii Conway; Conway 1976a, 1976b, Sanders and Theriot 1980, Charudattan 1984, Freeman and Charudattan 1984, Martyn 1985, Charudattan 1996, Tessmann et al. 2001), Myrothecium roridum Tode ex Fr. (Okunowo et al. 2008). Pathogens indigenous to a region, and those that cause endemic diseases, are ideal candidates for development as non-classical (augmentative or inundative) biological control agents (Cuda et al. 2008). While there has been considerable research on arthropod biological control agents of water hyacinth in South Africa (Coetzee et al. 2011), the role of indigenous fungal pathogens in the control of water hyacinth has not been studied. The international mycoherbicide programme for water hyacinth control in Africa (IMPECCA) was established to provide technical assistance to national programmes across the African continent for the African Journal of Aquatic Science is co-published by NISC (Pty) Ltd and Taylor & Francis Ray and Hill 324 Downloaded by [P Ray] at 19:00 16 November 2012 development of a mycoherbicide to control water hyacinth (Bateman 2001). The programme involved the International Institute of Tropical Agriculture (IITA), Benin, the University of Mansoura, Egypt; the Agricultural Research Council– Plant Protection Research Institute (ARC–PPRI), South Africa; the Seed Services of the Department of Research and Specialist Services (DRSS), Zimbabwe; the Danish Institute of Agricultural Sciences (DIAS), Denmark; and the CABI Bioscience, UK. The major objective of this collaboration was to survey, collect and isolate fungal pathogens from water hyacinth and to study their potential as mycoherbicides against the weed. The promising pathogens that were shortlisted for potential development as mycoherbicides from around the continent, according to order of preference, were A. eichhorniae, A. zonatum, C. piaropi, R. solani, A. alternata and M. roridum (Bateman 2001). However, the IMPECCA project was terminated without the discovery of a suitable fungus for mycoherbicide development, and not much work has yet been undertaken in South Africa. 0 With the growing interest in the use of fungi for weed control, the aim of this study was to survey for fungal pathogens on water hyacinth in South Africa and to test their pathogenicity against the weed under controlled conditions. Materials and methods Survey for collection of fungi associated with water hyacinth Water hyacinth leaves with disease symptoms, suspected to be damaged by fungal pathogens, were collected from various water hyacinth infested sites around South Africa during 2010 and 2011 (Figure 1, Table 1) covering wide geographical areas of the country with different climatic conditions. Plant and leaf specimens with disease symptoms were wrapped in layers of dry paper towelling to enable absorbance of their moisture content to prevent secondary microbial growth, kept in paper bags and sent to the laboratory for isolation and pathogenicity testing. 400 800 km AFRICA South Africa Collection sites 24° S Water hyacinth sites Johannesburg 26° S Durban 30° S ATLANTIC OCEAN East London Cape Town 34° S Port Elizabeth INDIAN OCEAN 18° E 24° E 30° E Figure 1: Areas of South Africa infested by Eichhornia crassipes, and the collection locations of diseased plant parts from which phytopathogens were isolated Downloaded by [P Ray] at 19:00 16 November 2012 African Journal of Aquatic Science 2012, 37(3): 323–331 325 Isolation of pathogens The diseased leaves were washed thoroughly in running tap water to remove unwanted soil particles. The isolation of the potential fungal agents was performed by transferring disease marks on the leaves to media plates. About 2 mm2 cross-sectional segments of the leaves and petiole were cut from the margins of necrotic or chlorotic lesions and surface-sterilised by sequential immersion in 70% ethyl alcohol to improve sodium hypochlorite penetration, 10% sodium hypochlorite (v/v) to eliminate contaminating superficial propagules and, finally, thrice in sterile distilled water to eliminate traces of the disinfectants used. The medium for the isolations was rose bengal chloramphenicol agar (Biolab, Merck, Gauteng, RSA) and potato dextrose agar (PDA) (Biolab, Merck, Gauteng, RSA) supplemented with chloramphenicol (10% w/v) in Petri dishes, and incubated under sterile conditions at 27 °C. Isolations were also attempted on water hyacinth extract dextrose agar (WhDA) plates (200 g freshwater hyacinth leaves; 15 g dextrose [Merck Chemicals Pty Ltd, Gauteng, RSA]; 20 g agar-agar [Biolab, Merck, Gauteng, RSA]; and 1 000 ml distilled water). The WhDA was prepared as follows: freshwater hyacinth leaves were washed in running tap water and then in distilled water. They were chopped into small fragments and boiled for 20–25 min in 500 ml distilled water and filtered through cheesecloth for the collection of extract. Dextrose and agar-agar were added to this extract and boiled until transparent. Culturing and sub-culturing The fungal species isolated from water hyacinth were purified by streak-plate and sub-culturing techniques (Agarwal and Hasija 1986). The growing edges of fungal colonies isolated were transferred to malt yeast extract agar (MEA) (Biolab, Merck, Gauteng, RSA) plates. Fungi were transferred serially until pure cultures were obtained. Cultures that appeared contaminated with other fungus were sub-cultured and purified. Table 1: Water hyacinth infested sites surveyed in 2010–2011 for the collection of fungi Frequency (%)  Number of isolates in a genus  100 Total no. of isolates Site Site no. 1 Goudini Road, Worcester, Western Cape 2 Kluitjieskraal, Tulbagh, Western Cape 3 Kubusi River, Stutterheim, Eastern Cape 4 Lake Nsezi, Empangeni, KwaZuluNatal 5 Muldersdrift, Johannesburg, Gauteng 6 Nahoon River, East London, Eastern Cape 7 Nseleni River, Empangeni, KwaZulu-Natal 8 Princess Vlei, Cape Town, Western Cape 9 PPRI, Pretoria, Gauteng 10 Rhodes University, Grahamstown, Eastern Cape 11 Rietondale, Pretoria, Gauteng 12 Swartkops River, Port Elizabeth, Eastern Cape 13 Tongaat Sugar Estates, Tongaat, KwaZulu-Natal 14 Wriggleswade Dam, Stutterheim, Eastern Cape Latitude (°S) 33.64420 Longitude (°E) 19.29980 33.43628 19.17581 32.59257 27.42184 28.73215 31.98987 26.03555 27.84850 32.97392 27.92570 28.74739 31.96890 34.04351 18.48781 25.67732 33.31022 28.28601 26.51847 25.73142 33.99525 28.22393 25.53375 29.27172 31.35584 32.55905 27.49288 Identification and maintenance of isolates The purified cultures of all the isolates were numbered and multiplied on PDA plates. The stock cultures of the microorganisms were maintained on PDA slants supplemented with 10% WhDA and MEA media and stored at 4–70 °C in refrigerator. The other slants were kept in the BOD incubator at 27 ± 10 °C and routinely transferred into fresh slants. The fungi were identified on the basis of their morphological growth characteristics, sporulation, conidial measurement and ability to produce pigmentation on growth media, using various available literature (Gilman 1959, Barnett 1960, Ellis 1971, 1976, Holliday 1993, Domsch et al. 2007). Frequency of occurrence of fungal isolates The fungal genera isolated from various water hyacinth infested sites were counted for their frequency of occurrence as compared to the other genera. The isolation frequency of each genus was expressed as the percentage of the total number of fungal isolates representing a given genus using the formula: Pathogenicity The fungal isolates were tested for their ability to infect water hyacinth plants in vitro. The pathogenicity trials were undertaken in two steps. In the first trial a 2 mm disc of fungus with its medium was cut from actively growing culture in Petri plate and placed on a piece of water hyacinth leaf on moist filter paper in a Petri plate and incubated in a walk-in BOD incubator at 26 ± 2 °C under a photoperiod of 14:10 h (L:D) for for 5–7 days. The leaf discs were observed for the development of disease symptoms. The fungi which caused disease on water hyacinth in this first round went for a second round of assessment, i.e. whole plant bioassay (Table 2). In the second trial, young water hyacinth plants were collected from local water bodies and grown in water tanks in a polyethylene tunnel at Rhodes University, Grahamstown, South Africa. They were fertilised with 15-3-12 N:P:K slow-release fertiliser (Multicote 8, Haifa Chemicals Israel, RSA [Pvt.] Ltd). A commercial iron chelate (13% Fe) was also added to the water at a concentration of 2 g per 23 litres of water. They were sprayed with insecticide, Malathion (Kombat [Pty] Ltd), to keep them free from insect infestation, as and when required. For the pathogenicity test healthy individual plants were collected from these tanks, washed thoroughly in running tap water followed by sterile distilled water. They were wiped with a cotton swab dipped in 70% alcohol and placed in small tubs containing tap water, Ray and Hill 326 Downloaded by [P Ray] at 19:00 16 November 2012 Table 2: Fungi isolated from water hyacinth in South Africa Fungal isolate Location of isolation * Acremonium (Cephalosporium) zonatum (Sawada) Gams Acremonium sp. Alternaria alternata (Fr.) Keissler Alternaria eichhorniae Nag Raj & Ponnappa 12, 13 6, 10, 12 3 1, 3, 7, 10, 12 Alternaria tenuissima (Nees ex Fr.) Wiltshire Alternaria sp. Bipolaris hawaiiensis (M.B. Ellis) Uchida & Aragaki Chaetomium sp. Chrysosporium merdarium var. roseum W. Gams Cladosporium sp. Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert & Gams Colletotrichum sp. Cylindrocladium sp. Epicoccum nigrum Link Eurotium sp. Exserohilum rostratum (Drechsler) K.J. Leonard & Suggs Fusarium avenaceum (Fr.) Sacc. Fusarium equiseti (Corda) Saccardo Fusarium moniliforme Sheldon Fusarium oxysporum (Schlecht) Fusarium solani (Mart.) Sacc. Fusarium sp. Gloeosporium sp. Macrophoma sp. Myrothecium roridum Tode ex Fr. 10 14 3 11 7 7, 10 7 6 7 1, 7 14 9 9 6 Nigrospora sp. Periconia sp. Pestalotia sp. Phoma sp. Pythium sp. Scopulariopsis sp. Stemphylium sp. Trichothecium sp. Ulocladium sp. 4, 7, 13 1 7, 13 7, 10 10 12 2 13 10 10 3, 4, 5, 6, 7, 10, 11 1 1 11 3, 9, 10, 11, 13, 14 4 Countries from which the isolate has previously been reported Australia, India, Mexico, Nigeria, Pakistan, Panama, Uganda, USA, Sudan Peru Australia, Bangladesh, Egypt, Ethiopia, India, Mexico Bangladesh, Egypt, Ghana India, Indonesia, Kenya, Nigeria, Thailand, USA, Uganda, South Africa, Zimbabwe Hong Kong Mexico, Sri Lanka, USA Mexico Egypt ** India, USA ** ** ** India, Mexico ** ** ** Ethiopia, India, Sudan India, Sudan India, Australia India, Australia India, Mexico, Nigeria, Peru, Sri Lanka, Uganda ** India Burma, India, Indonesia, Malaysia, Mexico, Nigeria, Philippines, Sri Lanka, Thailand, Uganda Mexico, USA Mexico, USA India, Mexico Brazil, India, Peru, USA, Uganda Ethiopia, India, USA ** Ethiopia, Mexico, USA ** Egypt * See site numbers in Table 1 ** Possible new host records supplemented with fertiliser when required. Before inoculation, some of the leaves from each plant were injured on their upper surface by making cut marks with a sterile scalpel blade. The fungi that were to be tested for their pathogenicity to water hyacinth were grown on PDA plates and incubated in a walk-in BOD incubator. The conidial/mycelial suspensions were prepared from 21-day-old culture in sterile distilled water. The conidial and mycelial mass was harvested by flooding the plates with sterile distilled water and then scraping the mass with a sterilised spatula. To this, Tween 20 (oxysorbic polyxyethylene sorbitan monoleate) was added as surfactant at the rate of 0.05 ml per 50 ml of spore suspension. Spore/mycelial suspensions were applied on water hyacinth until runoff. Control plants were sprayed with sterile distilled water containing Tween 20. These plants were then kept in the walk-in incubator at 27 °C and 80% relative humidity and 12 h photoperiod. The plants were individually enclosed in plastic bags to create a dew effect that was conducive to fungal growth. The disease severity was assessed visually every 24 h for a period of 25 days and the intensity of infection was measured using a score chart framed by Freeman and Charudattan (1984) and designated as: – (no symptom: healthy plant), + (mild symptom: plant showing slight symptoms up to 15% of leaf area), ++ (moderate symptom: plant showing definite bigger patches of diseased areas from 16% to 59% of leaf area) and +++ (severe symptom: enlarged lesions covering 60–100% of leaf area). Results Samples of diseased water hyacinth leaves collected from the field had variable diseased lesions including leaf spots, necrotic flecks, leaf blights, petiole rot, zonate lesions of various shapes and sizes and dieback symptoms Downloaded by [P Ray] at 19:00 16 November 2012 African Journal of Aquatic Science 2012, 37(3): 323–331 327 (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 2: Diseased Eichhornia crassipes leaves affected by various fungal species: (a) Fusarium oxysporum, (b) Acremonium zonatum and F. solani, (c) Alternaria alternata, (d) and (e) A. zonatum, (f) Alternaria eichhorniae, (g) Bipolaris hawaiiensis, (h) Ulocladium sp., (i) A. eichhorniae (Figure 2). About 250 fungal isolates belonging to more than 25 genera were purified from these diseased plant parts (Table 2). About 150 isolates were eliminated from further consideration because they were either contaminated, failed to grow, or belonged to the non-pathogenic and non-sporulating mycelia sterilia group. Several isolates showing rapid growth rate on PDA plates, mostly those of Aspergillus, Penicillium and Trichoderma, were excluded from further consideration after their initial isolation. Alternaria Nees was the commonest genus, with more than 30 isolates including three species, A. eichhorniae, A. alternata and A. tenuissima with a frequency of occurrence of 12.4% (Figure 3). This was followed by Cladosporium Link with 13 isolates (5.2% occurrence frequency) and Acremonium Link with 12 isolates (4.8% occurrence frequency). There were 10 isolates of Fusarium Link belonging to five species (F. avenaceum (Fr.) Sacc., F. oxysporum (Schlecht), F. equiseti (Corda) Saccardo, F. solani (Mart.) Sacc. and F. moniliforme Sheldon) with a frequency of occurrence of 3.6%. The pathogens that caused disease to water hyacinth leaf pieces in the first trial of pathogenicity testing in the Petri plate bioassay were further subjected to screening for their pathogenicity and damage to water hyacinth (Table 3) by whole plant bioassay. The appearance of symptoms on the leaves started within 3–6 days of application of inoculum in some of the viable isolates. These included five isolates of A. eichhorniae, three of A. zonatum and Fusarium spp. each, and one of Bipolaris hawaiiensis (M.B. Ellis) Uchida and Aragaki, M. roridum and Ulocladium Preiss sp. each. They were found to be highly virulent and severely damaging to the inoculated water hyacinth leaves. Two isolates of A. eichhorniae caused a rapid rate of infection and colonisation on the host plant, especially on the older leaves. In spite of the severe infection, in all cases new leaves continued to emerge on most of the plants and several of the plants survived. For example, one of the isolates of F. oxysporum caused about 90% damage to water hyacinth by the 30th day of application, yet on Ray and Hill 328 60 FREQUENCY (%) 50 40 30 20 Downloaded by [P Ray] at 19:00 16 November 2012 10 . . . . . . . . . . . . . . . . . . . . . . . . . i sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp sp ng s s s a a a a a a i i i i i m s m um um fu m um um y um um um um um um um m um or n m r t r m u u u u h h i i i i i i i a la p li lo o il ti o c le ri ri o ri ri p on tern ipo tom po po stac tric lad oc roh uro sa po ph hec ros ric sta Ph yth rio hy hec lad teri p a c c c t e t u s m s s P r o l l i e s o B e g r o E F eo c o o do n et P P e u m ho Ulo d/ ro Ni re A l dr Ep xs ha s op Ste ric ie lo Ma My Ac C hry Cla Clo ol lin c E T tif G C y S C n C de ni U Figure 3: Percentage frequency of occurrence of different fungal genera found on water hyacinth in South Africa in 2010–2011 the 45th day new leaves were observed to emerge, thus compensating for the damage caused by the fungi earlier. Discussion These surveys revealed that there is a rich diversity of fungal pathogens associated with water hyacinth in South Africa. Several of these mycobiota share a common link with those recorded in the centre of origin of the weed, the Amazon River Basin (Evans and Reeder 2001) and other countries where water hyacinth has been reported as a major weed. Several of these pathogens possibly co-evolved with the host plant and were spread to different parts of the world with the weed itself. However, the pathogenicity of the pathogens was variable. For example, highly virulent isolates of A. eichhorniae have been reported from Egypt (Shabana et al. 1995a, 1995b) and India (Nag Raj and Ponnappa 1970), while those previously reported from the USA (Freeman et al. 1974) and South Africa (Morris et al. 1999) have proved only weakly pathogenic to water hyacinth. Cercospora piaropi was reported on water hyacinth in South Africa (Morris 1990) and was reported to cause a severe decline of plants on a small farm dam in Mpumalanga province. The fungus was also imported from Florida, USA, and introduced into South Africa in 1988 (Morris et al. 1999). But, during the present study, it was not isolated in this country. Among the pathogens collected, isolates of A. eichhorniae, A. zonatum, B. hawaiiensis, F. oxysporum, F. solani, M. roridum, Ulocladium sp. and two isolates of Fusarium sp. appeared the most potent. Alternaria eichhorniae has been extensively studied for biocontrol potential against water hyacinth (Nag Raj and Ponnappa 1970, Shabana 1997, Shabana et al. 2000). It was observed to be host-specific to water hyacinth (Nag Raj and Ponnappa, 1970, Shabana et al. 1995a) and capable of severely damaging and suppressing this weed (Shabana et al. 1995a, 1995b, 1995c). However, one of the major obstacles to the use of A. eichhorniae as a mycoherbicide for water hyacinth is its requirement for at least 10 h of dew to allow the applied inoculum to germinate and infect and, to an extent, to colonise the weed (Shabana et al. 1995a). Rintz (1973) undertook extensive studies on A. zonatum from the biocontrol perspective and showed that the pathogen did not seem capable of killing water hyacinth, or of seriously hindering their prolific growth in the USA. During the present study, isolates of A. zonatum appeared substantially effective under greenhouse conditions in this study, but further studies would be needed to determine its potential as a biological control agent in the field. Several authors (Ponnappa 1970, Hettiarachchi et al. 1983, Liyanage and Gunasekera 1989, Okunowo et al. 2010) have also reported the potential of M. roridum as a biological control agent. While the isolates of B. hawaiiensis, F. oxysporum, F. solani and Ulocladium appear to have good potential, they still need to be evaluated for host specificity, virulence, and potential under various climatic conditions. Although these fungal pathogens have been found to be present on water hyacinth, not much work has been done to evaluate their biocontrol potential in South Africa. There are several difficulties associated with the use of fungi in the biological control of water hyacinth (Zorner et al. 1993, Boyetchko and Peng 2004); yet, with more studies, the use of phytopathogenic fungi as biocontrol agents could be valuable as these pathogens can cause a significant reduction in water hyacinth biomass, especially following natural disease outbreaks, after insect attacks, or when used as inundative bioherbicide agents. They can be used to manage invasive weeds in natural areas and in situations where non-chemical alternatives to weed control African Journal of Aquatic Science 2012, 37(3): 323–331 329 Downloaded by [P Ray] at 19:00 16 November 2012 Table 3: Evaluation of the impact of various fungi on water hyacinth Isolate no. Fungal isolate P-37 P-39 P-112 P-117 P-53 P-59 P-77 P-72 P-30 P-50 P-67 P-75 P-90 P-95 P-99 P-18 P-31 P-44 P-71 P-100 P-38 P-47 P-56 P-62 P-46 P-115 P-134 P-113 P-116 P-104 P-23 P-107 P-113 P-62 P-64 P-65 P-4 P-6 P-2 P-12 P-49 P-35 P-43 P-72 Acremonium zonatum Acremonium zonatum Acremonium zonatum Acremonium zonatum Acremonium sp. Acremonium sp. Acremonium sp. Alternaria alternata Alternaria eichhorniae Alternaria eichhorniae Alternaria eichhorniae Alternaria eichhorniae Alternaria eichhorniae Alternaria eichhorniae Alternaria eichhorniae Alternaria tenuissima Alternaria sp. Alternaria sp. Alternaria sp. Bipolaris hawaiiensis Cladosporium sp. Cladosporium sp. Clonostachys rosea Colletotrichum sp. Epicoccum nigrum Exserohilum rostratum Fusarium avenaceum Fusarium oxysporum Fusarium oxysporum Fusarium solani Fusarium sp. Fusarium sp. Fusarium sp. Gloeosporium sp. Macrophoma sp. Myrothecium roridum Nigrospora sp. Nigrospora sp. Pestalotia sp. Phoma sp. Phoma sp. Trichothecium sp. Trichothecium sp. Ulocladium sp. Intensity of infection on water hyacinth* +++ ++ +++ +++ – + ++ ++ ++ +++ ++ +++ +++ +++ +++ – + + + +++ – – + – – + + ++ +++ +++ – +++ +++ – – +++ + – + + – – – +++ Disease symptoms on leaves Zonate leaf spots Leaf spots Zonate leaf spots Zonate leaf spots No symptoms Leaf spots Leaf spots Leaf spot/blight Leaf spot Leaf spot/blight Leaf spots Leaf spot/blight Leaf spot/blight Leaf spot/blight Leaf spot/blight No symptoms Leaf spots Leaf spots Leaf spots Leaf spots No symptoms No symptoms Leaf spot No symptoms No symptoms Leaf spot Leaf spots Leaf spots Leaf spots Leaf spots No symptoms Leaf spot/blight Leaf spot/blight No symptoms No symptoms Leaf spots Chlorotic streaking No symptoms Leaf spots Leaf necrosis No symptoms No symptoms No symptoms Leaf spots * Intensity of infection (see: Methods: pathogenicity): – = no symptom, + = mild symptom, ++ = moderate symptom, +++ = severe symptom are needed. Although bioherbicides have been used as the sole option for the management of certain weeds in several cases (Daniel et al. 1973, Browers 1986, Kenney 1986, Riddings 1986, Mortensen 1988), for weeds like water hyacinth, they are likely to need to be supplemented with other control options such as in combination with different fungal pathogens (den Breeyen 1998, Ray et al. 2008b), the release of insect biocontrol agents (Charudattan et al. 1978, Denoth et al. 2002, Moran 2005, Yamoah et al. 2011) or used as a major supplement to low doses of conventional chemical herbicides (Charudattan 1986, Shearer and Nelson 2002, Nelson and Shearer 2005). The present study was carried out indoors in controlled experimental conditions. A follow-up study is needed that uses the priority pathogens identified in this paper, but in open, field-type environments. 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