This art icle was downloaded by: [ 196.45.51.39] On: 23 July 2014, At : 03: 37 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 Biocontrol Science and Technology 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/ cbst 20 Occurrence and effectiveness of an indigenous strain of Myrothecium roridum Tode: Fries as a bioherbicide for water hyacinth (Eichhornia crassipes) in Nigeria ab a Wahab O. Okunowo , Akinniyi A. Osunt oki , Adedot un A. c Adekunle & George O. Gbenle a a Depart ment of Biochemist ry, College of Medicine, Universit y of Lagos, Lagos St at e, Nigeria b Depart ment of Medicinal Chemist ry, College of Pharmacy, Universit y of Minnesot a Minneapolis, MN, USA c Depart ment of Bot any, Facult y of Science, Universit y of Lagos, Lagos St at e, Nigeria Accept ed aut hor version post ed online: 02 Sep 2013. Published online: 04 Oct 2013. To cite this article: Wahab O. Okunowo, Akinniyi A. Osunt oki, Adedot un A. Adekunle & George O. Gbenle (2013) Occurrence and ef f ect iveness of an indigenous st rain of Myrot hecium roridum Tode: Fries as a bioherbicide f or wat er hyacint h (Eichhornia crassipes) in Nigeria, Biocont rol Science and Technology, 23: 12, 1387-1401, DOI: 10. 1080/ 09583157. 2013. 839981 To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 09583157. 2013. 839981 PLEASE SCROLL DOWN FOR ARTI CLE Taylor & Francis m akes every effort t o ensure t he accuracy of all t he inform at ion ( t he “ Cont ent ” ) cont ained in t he publicat ions on our plat form . However, Taylor & Francis, our agent s, and our licensors m ake no represent at ions or warrant ies what soever as t o t he accuracy, com plet eness, or suit abilit y for any purpose of t he Cont ent . Any opinions and views expressed in t his publicat ion are t he opinions and views of t he aut hors, and are not t he views of or endorsed by Taylor & Francis. 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Term s & Condit ions of access and use can be found at ht t p: / / www.t andfonline.com / page/ t erm sand- condit ions Biocontrol Science and Technology, 2013 Vol. 23, No. 12, 1387–1401, http://dx.doi.org/10.1080/09583157.2013.839981 RESEARCH ARTICLE Occurrence and effectiveness of an indigenous strain of Myrothecium roridum Tode: Fries as a bioherbicide for water hyacinth (Eichhornia crassipes) in Nigeria Wahab O. Okunowoa,b*, Akinniyi A. Osuntokia, Adedotun A. Adekunlec and George O. Gbenlea a Downloaded by [196.45.51.39] at 03:37 23 July 2014 Department of Biochemistry, College of Medicine, University of Lagos, Lagos State, Nigeria; b Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota Minneapolis, MN, USA; cDepartment of Botany, Faculty of Science, University of Lagos, Lagos State, Nigeria (Received 27 May 2013; returned 25 June 2013; accepted 27 August 2013) In a study to isolate fungal pathogens with potential for the biocontrol of water hyacinth (Eichhornia crassipes), some lakes in the Lagos State and its environs, Nigeria, were surveyed for diseased water hyacinth (E. crassipes). The fungi present in the diseased tissue were isolated and identified as: Aspergillus niger, Aspergillus flavus, Penicillium sp., Curvularia pallescens, Fusarium solani and Myrothecium roridum. The pathogenicity of isolates of these fungi on fresh, non-diseased water hyacinth plants was investigated. Myrothecium was the only species capable of inducing disease symptoms. Necrosis was observed on water hyacinth leaves three days post inoculation (DPI) with M. roridum (1 × 106 spores/ml). The leaves and the petioles were withered at the end of day 24, and the disease incidence and disease severity were 100% and 8.67%, respectively. Molecular analysis of the internal transcribed spacer rDNA of the M. roridum isolate from water hyacinth showed >98% homology to authenticated sequences of M. roridum. The isolate, deposited at the International Mycological Institute, UK, as M. roridum Tode: Fries (IMI 394934), possesses the level of virulence needed in a potential mycoherbicide for use in the management of water hyacinth. Keywords: biocontrol; mycoherbicide; fungi; pathogen 1. Introduction Humans have facilitated the spread of water hyacinth, Eichhornia crassipes (Marts.) Solms-Laubach, from its native environment in South America to many regions throughout the world because of its attractive flowers. It made its entry into Nigerian waters via the Southwestern coastal border of Badagry around 1984 (Oso, 1988). Water hyacinth forms dense impenetrable mats that impede the recreational use of water and economic activities such as agricultural irrigation, navigation, fishing and power generation (loss of electricity production) (Mailu, 2001). These mats competitively exclude native aquatic plants and create good conditions for breeding disease vectors, particularly mosquitoes (Harley, 1990; Center, Hill, *Corresponding author. Email: modelprof@yahoo.com © 2013 Taylor & Francis Downloaded by [196.45.51.39] at 03:37 23 July 2014 1388 W.O. Okunowo et al. Cordo, & Julien, 2002). The losses caused by the weed in the several key sectors of some African countries is estimated to be in the order of billions of dollars (Mailu, 2001). The negative socio-economic and environmental impacts of this weed in many areas of the world are well documented in the literature (Mailu, 2001; Schmitz et al., 1993; Center et al., 2002). The indirect costs are enormous. Various control measures such as manual, mechanical, chemical and biological control are employed to check this water hyacinth. Some of these methods are, however, expensive and not environment-friendly. The use of conventional control measures such as mechanical removal, chemical herbicides and classical biological control using herbivorous insects are not entirely adequate and are probably expensive measures to apply on a large scale (Bateman, 2001). Biocontrol involves the use of host-specific natural enemies to minimise the population of a target pest. Several fungi and insects have been reported as control agents for aquatic weeds such as water hyacinth (Bateman, 2001; Charudattan, 2001, 1997; Coetzee, Hill, Julien, Center, & Cordo, 2009; Venter, Hill, Hutchinson, & Ripley, 2013). All biological control agents are specific on the target weed, generally persist at the site of infestation and tend to be self-regulating. Therefore, biological control is considered to be environmentally safe. Several plant pathogens have been tested and developed as biocontrol agents for large scale field application and over 15 have been used for biological control of weeds worldwide (Evans & Reeder, 2001). Several pathogens have also been tested for water hyacinth control though no commercial mycoherbicide was eventually developed (Dagno, Lahlali, Diourte, & Jijakli, 2011; El-Morsy, 2004; Praveena, Naseema, & George, 2007; Shabana & Mohamed, 2005; Tessmann, Charudattan, & Preston, 2008). However, the success of a fungal pathogen used as a biocontrol agent is influenced by environmental factors (Kirkpatrick, Templeton, TeBeest, & Smith Jr, 1982; TeBeest & Templeton, 1985; Walker, 1981), and one of the goals of biocontrol strategies is that potential biocontrol agents of pests should be isolated and studied in the region of the origin where the target organisms were suppressed naturally (Hong, Ryu, Hyun, Uhm, & Kim, 2002). This work reported here was carried out to survey, isolate, evaluate and identify an indigenous fungal agent which is biologically active under Nigerian climatic conditions for use locally and regionally as a biocontrol agent for water hyacinth. 2. Materials and methods 2.1. Survey for pathogens Field trips were undertaken to observe and examine waterways and lagoons of Badagry, Mile 2, Lagos and Ogun River (Isheri) (Figure 1) and to collect fungal pathogens from diseased water hyacinth. Sampling was done randomly at each sampling station: Lagos Lagoon, Mile 2, Ogun River and Badagry creeks using a motorised canoe at intervals of three months for a period of three years to collect fungal pathogens that attack the plant at various seasons of the year. The total area surveyed was 125 km2, 44 km2, 300 km2 and 45 km2 in Badagry, Mile 2, Lagos and Ogun River, respectively. Biocontrol Science and Technology 1389 Downloaded by [196.45.51.39] at 03:37 23 July 2014 Figure 1. (Colour online) The lagoons and creeks of Lagos and its environs surveyed for diseased water hyacinth. 2.2. Fungal isolation from diseased water hyacinth leaves Two square millimetre pieces were cut from the margins of necrotic lesions on the diseased leaf sample. These were surface sterilised in a solution of 0.26% sodium hypochlorite solution for 1 min and rinsed thrice in sterile water to remove traces of the disinfectant (Jimenez & Charudattan, 1998). Similarly, other pieces cut from typical lesions were sterilised in a solution of 1.4% sodium hypochlorite as described above. Five leaf pieces were placed on potato dextrose agar (PDA) and tap water agar (TWA), each containing an antibiotic (ampicillin; 500 mg/l), in petri plates and incubated at 25°C, with a 12 h dark/light regime to stimulate sporulation. All emerging fungi were isolated in pure cultures by the single hyphal-tip technique (Jimenez & Charudattan, 1998). 2.3. Morphological identification of fungal strains The pure cultures obtained were subcultured on plates containing 2% Malt Agar, one plate of TWA containing a single piece of sterile wheat straw and one plate of PDA. Cultures in plates were grown for 14 days under black light (wavelength 300–380 nm; 12 h alternating cycles black light/darkness) at 22°C to induce sporulation. At the end of this period, squash mounts of sporulating material were stained with lactophenol stain and examined under a light microscope. The fungal pathogens were identified according to their morphological appearance on the plates and the characteristics of spores under the light microscope. The growth (average diameter) of some of the fungi was determined on six replicate plates of PDA and the results are presented as mean ± standard deviation (SD). All isolates were screened for their pathogenicity on fresh non-diseased water hyacinth plants. 2.4. Pathogenicity test Healthy water hyacinth plants were collected, washed with 0.26% sodium hypochlorite solution and rinsed three times to eliminate insect infestation. Three plants (each average height 60–70 cm) were maintained in 20 l pot (30 cm diameter by 30 cm depth) containing 8 l of 50% Hoaglands solution (Jimenez & Charudattan, 1998) and allowed to equilibrate in the solution for one week prior to inoculation Downloaded by [196.45.51.39] at 03:37 23 July 2014 1390 W.O. Okunowo et al. with the pathogen. Inoculum was formulated by harvesting fungal spores from PDA culture plates in sterile distilled water containing 0.1% v/v Tween 80 solution. Leaves and petioles of experimental plants were inoculated with 200 ml of 1 × 106 spores/ml spore suspension containing 0.1% v/v Tween 80 using a hand-held low-pressure atomiser at a distance of 20 cm from the plant. The fungus was sprayed until run off on the leaves and stem of the plants. This experiment was conducted in six replicate pots and in three different experiments A, B and C in the University of Lagos. Control plants were also set up in the three different experiments by spraying leaves and petioles with sterile distilled water containing Tween 80 (0.1% v/v). Inoculated and control plants were immediately covered with sterile polythene bags for 48 h to maintain high relative humidity. The plants were then left in the open experimental field under the conditions of average temperature ranging between 24°C and 31°C, relative humidity between 68% in the night and 86% in the day and at an average rainfall of 25 mm for the month. The average sunlight/intensity was 7 h per day. Plants were monitored at three-day intervals for symptoms development. The isolates were ranked on the basis of the severity of the disease inflicted. Disease severity was assessed according to Freeman and Charudattan (1984). Finally, the pathogens were reisolated and identified from the inoculated and dead plants as well as from the control plants to fulfil Koch’s postulates. 2.5. Host range examination Host range of the most pathogenic isolate from the pathogenicity trial was tested on several local and economically important agricultural crops under field conditions (described above). The plants were sprayed with 200 ml fungal suspension (1 × 106 spores/ml in 0.1% v/v Tween 80 solution) and monitored for about three weeks for disease development and host plant reactions. Three individual plants in each pot were examined in triplicate experimental pots. Disease symptom rating was assessed by visual examination as: − = not susceptible (leaves healthy, no disease symptom observed), + = slightly susceptible (scanty leaf spotting or slight chlorosis no necrosis), ++ = susceptible (leaf spots/leaf necrosis at 30–50% leaf is dead) and + + + = highly susceptible (severe leaf spotting/necrosis at >50% leaf is dead). 2.6. Molecular characterisation of the pathogenic isolate The pathogenic isolate which was tentatively identified as Myrothecium sp. was further characterised by the Centre for Agriculture and Bioscience International (CABI), Egham, Surrey, UK using standard molecular identification technique to analyse the ITS1 rDNA sequence. The sequence and the isolate were deposited, respectively, in the GenBank (accession no. GQ853401) and the CABI microbial collection (Deposit no. IMI 394934). 2.7. Blast and phylogenetic analysis of the isolate The ITS1 rDNA sequence of our isolate obtained from CABI was subjected to homology analysis against the holdings of the GenBank using the software BLASTN 2.2.28+ (Zhang, Schwartz, Wagner, & Miller, 2000) and the phylogenetic relationship among taxa (for >97% homology) were determined using the neighbour-joining method (Saitou & Nei, 1987). Biocontrol Science and Technology 1391 2.8. Data analysis The data presented in this study are the results obtained from six replicate determinations and are expressed as mean ± SD. To determine the reproducibility of the experimental results, disease progression between the different experiments were compared using the general linear model (GLM) regression analysis with days post inoculation (DPI) set as continuous predictor. The analyses were done at critical P value of 0.05 using the GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California USA). Downloaded by [196.45.51.39] at 03:37 23 July 2014 3. Results 3.1. Survey for pathogens The continuous survey of the water bodies over the period of investigation showed that water hyacinth was prevalent at different periods in the year, particularly in the rainy season between late May and late September and absent in the dry season. Initial cursory observations in the field revealed that there was little evidence of occurrence of fungal pathogens on water hyacinth except during September to November when plants with unique pattern of infection were found in two of the four sampling stations: Badagry Creeks (N6.41950° E2.86019°, N6.42066° E2.86630° and N6.41748° E2.87552°) and Ogun River (N6.64091° E3.8406°) (Figure 2A). The disease appeared as a leaf spot, with concentric rings rounded on the side facing the petiole and narrowing towards the laminar tip. Older leaf spots turned necrotic with dark brown margins, with the centre of the spot containing white and black fungal spores. The diameter of each spot appeared to be proportional to the age of the spot. The disease was easy to identify as brownish necrotic leaf blight, forming massive brownish patches on water hyacinth leaves in the field. The total area infected was 0.0396 km2 in Badagry and 0.0074 km2 in Ogun River. The fungal prevalence or number of infection was 3.17 × 10−2 % of the total number of plants in Badagry Creeks and 1.64 × 10−2 % of the total number of plants surveyed in Ogun River. There were no infection in Mile 2 and Lagos Lagoon. 3.2. Fungal isolation from diseased water hyacinth leaves Five different fungi (Fusarium sp., Aspergillus niger, Aspergillus flavus, Curvularia sp., Penicillium sp.) were isolated from water hyacinth leaf pieces sterilised with 0.26% sodium hypochlorite and plated on PDA. These fungi grew out within 24 h while a Myrothecium sp. appeared within 36 h. From leaf pieces sterilised with 1.4% sodium hypochlorite solution, Myrothecium sp. appeared conspicuously on day 3, while the other fungi appeared between days 5 and 6. Of these organisms, Myrothecium sp. occurred most frequently (45%) of total isolation (350) followed by Fusarium sp. (30%), Curvularia sp. (14.7%), Penicillium sp. (5%), A. niger (3.5%) and A. flavus (1.8%). The use of TWA medium yielded Myrothecium sp. after 24 h, although this appeared as transparent hyphae on the medium as compared to the fluffy, whitish, conspicuous appearance on PDA. The other fungi such as Fusarium sp. and Curvularia sp. on TWA were not noticeable until the fourth or fifth day. The growth of the other organisms appeared not to be well supported by TWA. W.O. Okunowo et al. Downloaded by [196.45.51.39] at 03:37 23 July 2014 1392 Figure 2. (Colour online) (A) Pattern of natural infection of water hyacinth by Myrothecium species at the survey site. (B) Morphological appearance of Myrothecium roridum (IMI 394934) on PDA plate. (C1 & C2) Photomicrograph of Myrothecium roridum (IMI 394934) conidia × 1000. (D–I) Disease progression in water hyacinth leaf post inoculation with Myrothecium roridum (IMI 394934) (1 × 106 spores/ml): (D) Day 0; (E) Day 3 (F) Day 6; (G) Day 9; (H) Day 12; (I) Day 21. 3.3. Fungal identification Cultures of one of the isolates on PDA plates (using an 8 mm diameter cork borer) reached 56 ± 3 mm diameter in six days at 25°C and appeared brownish-black with irregular border and concentric zones. Conidia were slightly curved, septate and the central cells were broader than the end cells. The isolate was identified and authenticated as Curvularia pallescens Boedijn by Dr Markus N. Thormann Biocontrol Science and Technology 1393 Downloaded by [196.45.51.39] at 03:37 23 July 2014 (Northern Forestry Centre, Natural Resources Canada, 5320-12251, Edmonton, AB T6H 3S5, Canada). Cultures of a second isolate on PDA reached 77.5 ± 4.2 mm diameter in six days at 25°C, slightly whitish at first and later turning pinkish in colour. Conidia were sickle shaped and septate. The isolate was authenticated as Fusarium solani by Prof. A.A. Adekunle (Botany and Microbiology Department, University of Lagos). The third isolate on PDA reached 77.13 ± 1.6 mm diameter in 14 days at 25°C. The isolate produced white, floccose colonies with sporodochia in dark green-toblack concentric rings (Figure 2B). Conidia were sub-hyaline and cylindrical with rounded ends (Figure 2C1 & C2). All characteristics were consistent with the description of Myrothecium roridum Tode ex Fr. (Ellis, 1971; Fitton & Holliday, 1970). This was authenticated as M. roridum and was given the accession number (IMI 394934) at the Centre for Agriculture and Bioscience International (CABI), Egham Surrey, UK. 3.4. Pathogenicity screening No disease symptoms were observed on water hyacinth plant infected with C. pallescens and F. solani 24 DPI. Of the six different fungal species tested for their ability to infect healthy water hyacinth plants in vitro, the result showed that M. roridum was the only candidate which infected and produced disease symptoms on water hyacinth leaves. The disease started as scanty patches which developed into pale-to-dark brown heavy necrotic spots on the leaves. The necrotic spots expanded in diameter between 5 and 10 mm. With disease progression, the necrotic spots coalesced and the necrotic area increased. The resultant effect was a decrease in the green leaf area and leaf death (Figure 2D–I). The symptom produced in the pathogenicity test was similar to that seen in the field (Figure 2A). The M. roridum isolate was ranked on the basis of the severity of the damage it caused (Table 1). The disease progression was monitored over time in terms of disease severity and disease incidence (Table 1). The disease incidence on day 4 was greater than 60% in experiments A–C, respectively, and 100% in these experiments on day 7. Similarly, the disease severity became prominent on day 4 in all experiments and the mean values were greater or equal to 2.60. The average disease severity on day 24 was maximum in experiment A and least in experiment C. However, regression analysis indicates that there was no significant difference in the rate of disease progression in all experiments (F2,27 = 0.95, P = 0.4). This is an indication that the result is reproducible. Based on the result obtained, the isolate of M. roridum (IMI 394934) was chosen for further study. 3.5. Host specificity test. The host range plant response to M. roridum showed that 74.19% of the test plants were not susceptible (Table 2). Slightly susceptible plants account for 16.13% and plants health status were not compromised. Duckweed was susceptible to the fungus resulting in necrosis and death of the plants. Water lettuce was highly susceptible. Water hyacinth was highly susceptible showing heavy leaf spotting and necrosis with more than 50% of leaf area coalescing with a resultant death of the plant in less than 21 days. 1394 W.O. Okunowo et al. Table 1. Disease incidence (Exp. A) DPI 0 3 4 5 7 10 11 14 17 20 24 Downloaded by [196.45.51.39] at 03:37 23 July 2014 Pathogenicity profile of Myrothecium roridum (IMI 394934) on water hyacinth. 0.00 0.00 83.33 100 100 100 100 100 100 100 100 Mean disease severity (Exp. A) 0.00 0.00 2.60 3.17 4.17 5.00 5.17 5.83 6.83 7.83 8.67 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.00 0.62 0.72 0.66 0.57 0.54 0.62 0.62 0.77 0.62 Disease incidence (Exp. B) 0.00 0.00 66.67 83.33 100 100 100 100 100 100 100 Mean disease severity (Exp. B) 0.00 0.00 2.75 3.00 4.00 4.67 4.67 5.53 5.94 6.67 7.67 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.00 0.45 0.52 0.57 0.51 0.51 0.47 0.54 0.52 0.62 Disease incidence (Exp. C) 0.00 0.00 66.67 83.33 100 100 100 100 100 100 100 Mean disease severity (Exp. C) 0.00 0.00 2.75 3.11 3.94 4.72 4.78 5.33 5.83 6.50 7.28 ± ± ± ± ± ± ± ± ± ± ± 0.00 0.00 0.47 0.62 0.50 0.50 0.60 0.83 0.93 0.87 0.75 Data represent mean ± SD of six replicate determinations. *P < 0.05 = Significant difference in data exist between different experiments (Exp. A–C) when subjected to GLM regression analysis with DPI set as continuous predictor. Data for control plants were excluded as disease symptoms were absent. DPI, days post inoculation; GLM, general linear model. Disease incidence = (number of leaves with disease symptoms/total number of leaves present on plants in six replicate pots) × 100. Disease severity keys: 0 1 2 3 4 5 6 7 8 9 = = = = = = = = = = no spots on lamina or petiole. 1–4 spots on lamina, no petiolar spotting. Less than 25% of lamina surface with spots, no coalescence or petiolar spotting. Less than 50% of laminar surface with spots, some coalescence, no petiolar spotting. Less than 50% of leaf surface with spots, coalescence, some tip dieback, and petiolar spots. Less than 50% of leaf surface with spots, coalescence, 10% tip dieback, and petiolar spotting. Less than 75% spots, coalescence, 30% tip dieback, and petiolar spotting. Greater than 75% spots, coalescence, 60% tip dieback, coaleascing spots on petiole. Dead lamina, petiole green, but heavily spotted. Dead lamina and petiole (submerged). 3.6. Molecular identification of the pathogenic isolate Results from CABI microbial identification service indicated that the morphology of the strain conforms in all respects to standard descriptions of Myrothecium species and the ITS1 rDNA sequence data of 533 base pairs revealed that it is a new strain of M. roridum (GenBank accession no. GQ853401). 3.7. Blast and phylogenetic analysis Sequence alignment of the ITS rDNA of the isolate IMI394934 did not produce any species with 100% homology (Table 3). The closest species which were from marine sources and agricultural crops were 99% homologous and differed by four base pairs. However, we could not find any sequence data for previously reported isolates of water hyacinth in the GenBank/alignment search. The strain is clustered with a number of other, previously described, M. roridum isolates (Figure 3). Moreso, this isolate was most related to a strain identified as 1395 Biocontrol Science and Technology Table 2. Plant response to Myrothecium roridum (IMI 394934) 21 days post inoculation. Downloaded by [196.45.51.39] at 03:37 23 July 2014 Plant family Amaranthaceae Amaranthaceae Anacardiaceae Apiaceae Araceae Araceae Araceae Arecaceae Asteraceae Asteraceae Brassicaceae Bromeliaceae Caricaceae Cucurbitaceae Dioscoreaceae Euphorbiaceae Euphorbiaceae Euphorbiaceae Fabaceae Fabaceae Hydrocharitaceae Musaceae Nymphaeaceae Poaceae Poaceae Pontederiaceae Solanaceae Solanaceae Solanaceae Sparrmanniaceae Xanthorrhoeaceae Botanical name Common name Host response/disease rating Amaranthus viridis Celosia argentea Mangifera indica Daucus carota Colocasia esculenta Lemna minor Pistia stratiotes Phoenix dactylifera Lactuca taraxacifolia Vernonia amygdalina Brassica oleracea Ananas comosus Carica papaya Citrullus lanatus Dioscorea alata Acalypha cordifolia Manihot esculenta Euphorbia milii Vigna unguiculata Arachis hypogaea Hydrilla verticilata Musa paradisiacal Nymphaea caerulea Zea mays Cymbopogun citrates Eichhornia crassipes Capsicum chinense Capsicum annuum Nicotiana tabacum Corchorus olitorius Aloe vera Green amaranth Plumed celosia Mango Carrot Cocoyam Duckweed Water lettuce Date palm Lettuce Bitter leaf Cabbage Pineapple Pawpaw Water melon Yam Acalypha Cassava Crown of thorn Beans Groundnut Hydrilla Banana Water lilly Corn Lemon grass Water hyacinth Red savina Chili pepper Tobacco Jute Aloe − − − − − ++ +++ − − + − − − − − − − − + + − − − − + +++ − − − + − Disease rating scale: − = not susceptible (leaves healthy, no disease symptom observed), + = slightly susceptible (scanty leaf spotting or slight chlorosis or no necrosis), ++ = susceptible (leaf spots/leaf necrosis at 30–50% leaf is dead), and + + + = highly susceptible (severe leaf spotting/necrosis at >50% leaf is dead). The fungal suspension was applied at 1 × 10−6 spore/ml in 0.1% v/v Tween 80 solution. M. carmichaelii isolate IMI 199044 (GenBank accession no. AY254150). Our isolate has been registered in the International Mycological Institute (IMI) Culture Collection Center (M. roridum IMI 394934). The molecular sequence data of the internal transcribed spacer regions ITS1, ITS2 and the 5.8s rRNA genomic region of the isolate has also been deposited in the GenBank (accession no. GQ853401). 4. Discussion The symptoms or diseased water hyacinth plants were peculiar to two of the sampled locations; Badagry Creeks (N6.41950° E2.86019°, N6.42066° E2.86630° and N6.41748° E2.87552°) and Ogun River (Isheri: N6.64091° E3.8406°). Previous explorative studies in Nigeria have shown the presence of some fungal isolates such 1396 W.O. Okunowo et al. Table 3. Alignment analysis of ITS rDNA sequence of Myrothecium roridum (IMI 394934) with the closest (≥97% homology) fungi in the holdings of GenBank. SN 1 Downloaded by [196.45.51.39] at 03:37 23 July 2014 2 3 4 5 6 7 8 9 10 11 12 13 14 GenBank accession numbers Homology percentage M. roridum (isolate IMI 394934) GQ853401 100 M. roridum (strain 784) M. carmichaelii (strain IMI 199044) M. roridum (strain 794) M. roridum (strain MA-73) M. roridum (strain 801) M. roridum (strain 802) M. roridum M. roridum (strain 782) M. roridum (strain CICR) M. roridum (strain CD08072303) Pteris ensiformis Myrothecium sp. (isolate HKB 34) M. roridum (strain DGM01) JF724157 AY254150 99 99 JF724158 JF724153 JF724151 JF724150 EF151002 JF724156 EU927366 GQ381291 AM920397 EF029818 JF343832 99 99 99 99 99 99 99 99 99 99 98 AJ302001 98 AJ301995 JF724155 GQ162434 GU722059 98 98 98 97 JF724152 AY254159 JQ081552 97 97 97 HQ637275 AJ302000 97 97 GQ921722 97 Sources Water hyacinth (Nigeria) Melon (Brazil) Marine (Spain) 15 Soybean leaf (Brazil) Soybean leaf (Brazil) Soybean root (Brazil) Soybean root (Brazil) Salvia sp. (USA) Melon root (Brazil) Cotton leaf (India) Bean (China) Young fronds (India) Marine sponge (USA) Hemionitis arifolia leaf (China) (Germany) 16 17 18 19 (Germany) Soybean leaf (Brazil) Tomato leaf (China) Surface dust (USA) 20 21 22 Soybean leaf (Brazil) Marine (Spain) Soil (Brazil) 23 24 Soil (China) (Germany) 25 Soil (Australia) Species M. roridum (strain BBA 71015 {CBS 212.92}) M. roridum (strain BBA 67679) M. roridum (strain 781) M. roridum (strain FQ07090401) Uncultured fungus (clone f4HSc41) M. roridum (strain MA-20) M. lachastrae (strain IMI 273160) Uncultured fungus (clone ASSA173) Myrothecium sp. (strain JZ-45) M. leucotrichum (strain BBA 71014 {CBS 131.64}) Uncultured fungus (clone RFLP type 6) E value = 0, for all sequences in the table. as Cercospora piaropi Tharp, Cladosporium oxysporum Berk. & Curt and Phyllosticta sp. on water hyacinth (Barreto & Evans, 1996). In this study, a different fungal species identified as M. roridum (IMI 394934) was obtained. Reports indicate that strains of this fungus have been isolated in India, Mexico, Philippines Thailand/ Burma (IMI 79771) and Malaysia (IMI 277583) (Charudattan, 2001; Evans & Reeder, 2001). However, to the best of our knowledge this is the first documented report of M. roridum isolation from water hyacinth in Nigeria. As a result of pathogenicity testing and on the basis of disease severity, M. roridum (IMI 394934) was found to be highly destructive on water hyacinth. Downloaded by [196.45.51.39] at 03:37 23 July 2014 Biocontrol Science and Technology 1397 Figure 3. Phylogenetic analysis of ITS rDNA sequence data of Myrothecium roridum IMI 394934 (gb/GQ853401) with 24 most homologous sequences available in the GenBank. Barreto and Evans (1996) and Charudattan (2001) reported that only a few fungi recorded on water hyacinth have been thoroughly tested and confirmed to be highly virulent pathogens. Of these fungi, Acremonium zonatum, Alternaria eichhornia and C. piaropi (= C. rodmanii), under experimental conditions have been shown as biocontrol agents effective against in water hyacinth (Charudattan, 2001). Also, Bateman (2001) reported some promising fungi as potential mycoherbicides for water hyacinth control in Africa. In order of potential utility based on the virulence, they include A. eichhorniae, A. zonatum, C. piaropi, Rhizoctonia solani, Alternaria alternata and M. roridum. However, the strain of M. roridum (IMI 394934) reported in this study appeared to show a greater disease incidence and disease severity than that reported for A. alternata (El-Morsy, Dohlob, & Hyde, 2006), since healthy water hyacinth used in this study died four weeks post inoculation with M. roridum (IMI 394934), taking into consideration that this study was carried out under different climatic and environmental conditions. C. pallescens and F. solani isolated in this study were not virulent and not considered potential candidates for water hyacinth control. This is in agreement with the previous reports from some other countries which also found these two organisms to be non-virulent fungi associated with water hyacinth (Barreto & Evans, 1996; El-Morsy et al., 2006). The basic sequence alignment analysis of the internal transcribed spacer of the isolate IMI 394934 (GenBank accession no. GQ853401) showed no strains with 100% homology; an indication that it is a new strain of M. roridum. However, it was Downloaded by [196.45.51.39] at 03:37 23 July 2014 1398 W.O. Okunowo et al. 99% homologous to a strain identified as the closely related species M. carmichaelii isolate IMI 199044 (GenBank accession no. AY254150). This suggests that the difference in homology is not sufficient to establish an unequivocal identification. The paucity in the sequence data of the previous isolates of water hyacinth in Sri Lanka; IMI 261802 (Hettiarachchi, Gunasekera, & Balasooriya, 1983), India (Ponnappa, 1970), Mexico/India/Philippines Thailand/Burma (IMI 79771) and Malaysia (IMI 277583) (Barreto & Evans, 1996; Evans & Reeder, 2001) made it impossible to compare our isolate or perform phylogenetic relationship studies among isolates of M. roridum pathogenic to water hyacinth. Phylogenetic studies have been done on Cercospora species pathogenic to water hyacinth (Tessmann, Charudattan, Kistler, & Rosskopf, 2001), such studies could offer some insights into biogeographic hypothesis of Myrothecium on water hyacinth. Our isolate was able to weakly infect bean and groundnut; however, it is not clear if the isolates from bean, soybean and other agricultural crops can infect or be pathogenic to water hyacinth. M. roridum has been previously reported as a pathogen of water hyacinth and some other host plants including some economically important crops (Fish, Bruton, & Popham, 2012; Gaikwad, 1988; Hettiarachchi et al., 1983; Ponnappa, 1970). The non-host nonspecificity was confirmed in this study by the ability to cause slight disease on bitter leaf, bean, groundnut, lemon grass and jute plants. The isolate studied caused no disease symptoms in corn unlike the report of Gaikwad (1988), this may be due to the differences in the source and origin of the isolates. Several studies indicate that the difference in the source or origin of microorganisms affects their performance (Anuna & Akpapunam, 1995; Anuna, Sokari, & Akpapunam, 1990; Okunowo & Osuntoki, 2007). We have previously reported its virulence on water lettuce (Okunowo, Osuntoki, & Adekunle, 2011). The efficacy of the fungus in the integrated management of water hyacinth is known to be enhanced by 2,4 D (Liyanage & Gunasekera, 1989). Hoagland, Weaver, and Boyette (2007) elucidated some possible strategies to reduce the non-target risk of a promising mycoherbicidal agent Myrothecium verrucaria which can be adapted to reduce the non-host specificity of M. roridum. Conclusively, this study has isolated and identified a Nigerian indigenous strain of M. roridum, which is highly virulent to water hyacinth. This M. roridum isolate has potential for application in the biocontrol of water hyacinth. However, since it is not host specific, future studies should include extensive host range tests and strategies to reduce its non-target risk. Acknowledgements The authors are grateful to Raghavan Charudattan (Emeritus Professor, Plant Pathology Department, University of Florida, Gainsville, Florida, USA) and Hamed K. Abbas (Research Plant Pathologist/Lead Scientist, Biocontrol of Pest Research Unit, USDA-ARS, NBCL, Stoneville, MS, USA) for their helpful discussions and review of the draft version of the manuscript. References Anuna, M. I., & Akpapunam, M. A. (1995). Quantitative analysis of alcohol types in pineapple (Ananas comosus) L (L). Merr.) wine fermented by two strains of Saccharomyces cerevisiae. Nigerian Food Journal, 13, 12–17. Downloaded by [196.45.51.39] at 03:37 23 July 2014 Biocontrol Science and Technology 1399 Anuna, M. I., Sokari, T. G., & Akpapunam, M. A. (1990). Effect of source of yeast (Saccharomyces spp.) on alcohol content and quality of pineapple (Ananas comosus) wine. Discovery and Innovation, 2, 80–84. Barreto, R. W., & Evans, H. C. (1996, January). Fungal pathogens of some Brazilian aquatic weeds and their potential use in biocontrol. Paper presented at the 9th international symposium on Biological Control of Weeds, Stellenbosch, South Africa. Bateman, R. (2001). IMPECCA: An international, collaborative program to investigate the development of a mycoherbicide for use against water hyacinth in Africa. In M. H. Julien, M. P. Hill, T. D. Center, & D. Jianqing (Eds.), Biological and integrated control of water hyacinth, Eichhornia crassipes. Proceedings of the second meeting of the Global Working Group for the biological and integrated control of water hyacinth (pp. 57–61), Beijing, China, October 9–12, 2000. ACIAR Proceedings No. 102. Center, T. D., Hill, M. P., Cordo, H., & Julien, M. H. (2002). Water hyacinth. In R. Van Driesche, B. Blossey, M. Hoddle, S. Lyon, & R. Reardon (Eds.), Biological control of invasive plants in the Eastern United States (413 p). Morgantown, WV: USDA Forest Service Publication Forest Health Technology Enterprise Team-2002-04. Retrieved from http://dnr.state.il.us/stewardship/cd/biocontrol/4WaterHyacinth.html. Charudattan, R. (2001). Biological control of water hyacinth by using pathogens: Opportunities, challenges, and recent developments. In M. H. Julien, M. P. Hill, T. D. Center, & D. Jianqing (Eds.), Biological and integrated control of water hyacinth, Eichhornia crassipes. Proceedings of the second meeting of the Global Working Group for the biological and integrated control of water hyacinth (pp. 21–28), Beijing, China, October 9–12, 2000, ACIAR Proceedings No. 102. Charudattan, R. (1997). Bioherbicides for the control of water hyacinth: Feasibility and needs. In: E. S. Delfosse & N. R. Spencer (Eds.), Proceedings of the international water hyacinth consortium. Held at the World Bank, March 18–19, 1997. Washington, DC: World Bank. Coetzee, J. A., Hill, M. P., Julien, M. H., Center, T. D., & Cordo, H. A. (2009). Eichhornia crassipes. In R. Muniappan, G. V. P. Reddy, A. Raman, & V. P. Gandhi (Eds.), Weed biological control with arthropods in the tropics (pp. 183–210). Cambridge: Cambridge University Press. Dagno, K., Lahlali, R., Diourté, M., & Jijakli, M. H. (2011). Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes). Journal of Applied Microbiology, 110, 521–528. doi:10.1111/j.1365-2672.2010.04908.x Ellis, M. B. (1971). Myrothecium. In: Dematiaceous hyphomycetes. Kew, Surrey, England: Commonwealth Mycological Institute CAB. El-Morsy, E. S. M. (2004). Evaluation of microfungi for the biological control water hyacinth in Egypt. Fungal Diversity, 16, 35–51. Retrieved from http://www.fungaldiversity.org/fdp/ sfdp/16-12.pdf El-Morsy, E. M., Dohlob, S. M., & Hyde, K. D. (2006). Diversity of Alternaria alternata a common destructive pathogen of Eichhornia crassipes in Egypt and its potential use in biological control. Fungal Divers, 23, 139–158. Retrieved from http://www.fungaldiversity. org/fdp/sfdp/23-8.pdf Evans, H., & Reeder, R. (2001). Fungi associated with Eichhornia crassipes (water hyacinth) in the upper Amazon basin and prospects for their use in biological control. In M. H. Julien, M. P. Hill, T. D. Center, & D. Jianqing (Eds.), Biological and integrated control of water hyacinth, Eichhornia crassipes. Proceedings of the second meeting of the Global Working Group for the biological and integrated control of water hyacinth (pp. 62–70), Beijing, China, October 9–12, 2000, ACIAR Proceedings No. 102. Fish, W. W., Bruton, B. D., & Popham, T. W. (2012). Cucurbit host range of Myrothecium roridum isolated from watermelon. American Journal of Plant Sciences, 3, 353–359. doi:10.4236/ajps.2012.33042 Fitton, M., & Holliday, P. (1970). No. 253 in: CMI descriptions of pathogenic fungi and bacteria. Great Britain: The Eastern Press. Freeman, T. E., & Charudattan, R. (1984). Cercospora rodmanii conway: A biocontrol agent for water hyacinth. Florida, FL: Agricultural Experiment Stations, Institute of Food and Agricultural Sciences, University of Florida Gainesville. Downloaded by [196.45.51.39] at 03:37 23 July 2014 1400 W.O. Okunowo et al. Gaikwad, S. J. (1988). Host range studies of Myrothecium roridum Tode ex Fr the causal organism of Myrothecium leaf spot disease of pearl millet. Punjabrao Krishi Vidyapeeth Research Journal, 12, 162–163. Harley, K. L. (1990). The role of biological control in the management of water hyacinth, Eichhornia crassipes. Biocontrol News and Information, 11, 11–22. Hettiarachchi, S., Gunasekera, S. A., & Balasooriya, I. (1983). Leaf spot diseases of water hyacinth in Sri Lanka. Journal of Aquatic Plant Management, 21, 62–65. Retrieved from http://www.apms.org/japm/vol21/v21p62.pdf Hoagland, R. E., Weaver, M. A., & Boyette, C. D. (2007). Myrothecium verrucaria fungus: A bioherbicide and strategies to reduce its non-target risks. Allelopathy Journal, 19, 179. Retrieved from http://www.ars.usda.gov/SP2UserFiles/Place/64022000/Publications/Boyette/Hoaglandetal.07AllelJ19-179-192.pdf Hong, Y.-K., Ryu, K.-L., Hyun, J.-N., Uhm, J.-Y., & Kim, S.-C. (2002). Distribution and changes in occurrence of fingerprint stem blight of Eleochariskuroguwai caused by Epicoccosorus nematosporus in Korea. The Plant Pathology Journal, 18, 152–155. doi:10.5423/PPJ.2002. 18.3.152 Jimenez, M. M., & Charudattan, R. (1998). Survey and evaluation of Mexican native fungi for potential biocontrol of water hyacinth. Journal of Aquatic Plant Management, 36, 145–148. Retrieved from https://www.apms.org/wp/wp-content/uploads/2012/10/v36p145.pdf Kirkpatrick, T. L., Templeton, E., TeBeest, D. O., & Smith Jr, R. J. (1982). Potential of Colletotrichum malvarum for biological control of prickly sida. Plant Disease, 66, 323–325. doi:10.1094/PD-66-323 Liyanage, N. P., & Gunasekera, S. A. (1989). Integration of Myrothecium roridum and 2, 4-D in water hyacinth management. Journal of Aquatic Plant Management, 27, 15–20. Retrieved from http://www.apms.org/japm/vol27/v27p15.pdf Mailu, A. M. (2001). Preliminary assessment of the social, economic and environmental impacts of water hyacinth in Lake Victoria Basin and status of control biological and integrated control of water hyacinth. In M. H. Julien, M. P. Hill, T. D. Center, & D. Jianqing (Eds.), Biological and integrated control of water hyacinth, Eichhornia crassipes. Proceedings of the second meeting of the Global Working Group for the biological and integrated control of water hyacinth (pp. 130–139), Beijing, China, October 9–12, 2000, ACIAR Proceedings No. 102. Okunowo, W. O., & Osuntoki, A. A. (2007). Quantitation of alcohols in orange wine fermented by four strains of yeast. The African Journal of Biotechnology, 1, 95–100. Retrieved from http://www.academicjournals.org/ajbr/PDF/Pdf2007/Nov/Okunowo%20and %20Osuntoki.pdf Okunowo, W. O., Osuntoki, A. A., & Adekunle, A. A. (2011). Myrothecium roridum Tode and its toxin shows potential for management of water lettuce. Phytopathology, 101, S131. Retrieved from http://www.apsnet.org/meetings/Documents/2011_Meeting_Abstracts/ a11ma768.htm Oso, B. A. (1988). Explorative studies for biological control agents of water hyacinth in Nigeria. In O. L. Oke, A. M. A. Imevbore, & A. F. Titilola (Eds.), Water hyacinth menace and resource. Proceedings of an international workshop held in Lagos Nigeria (pp. 129–136), August 7–12, 1988. Federal Ministry of Science and Technology Publication. Ponnappa, K. M. (1970). On the pathogenicity of Myrothecium roridum–Eichhornia crassipes isolate. Hyacinth Control Journal, 8, 18–20. Praveena, R., Naseema, A., & George, S. (2007). Effect of herbicides on Fusarium pallidoroseum – A potential biocontrol agent of water hyacinth [Eichhornia crassipes (Mart.) Solms]. Journal of Tropical Agriculture, 45, 55–57. Retrieved from http://www. jtropag.in/index.php/ojs/article/view/290/181 Saitou, N., & Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular biology and evolution, 4, 406–425. Retrieved from http://mbe. oxfordjournals.org/content/4/4/406.full.pdf Schmitz, D. C., Schardt, J. D., Leslie, A. J., Dray Jr, F. A., Osborne, J. A., & Nelson, B. V. (1993, October). The ecological impact and management history of three invasive alien aquatic plant species in Florida. Paper presented at the Biological Pollution: The Control and Impact of Invasive Exotic Species symposium held at Indianapolis, Indiana, IN, USA. Downloaded by [196.45.51.39] at 03:37 23 July 2014 Biocontrol Science and Technology 1401 Shabana, Y. M., & Mohamed, Z. A. (2005). Integrated control of water hyacinth with a mycoherbicide and a phenylpropanoid pathway inhibitor. Biocontrol Science and Technology, 15, 659–669. doi:10.1080/09583150500135842 TeBeest, D. O., & Templeton, G. E. (1985). Mycoherbicides: Progress in the biological control of weeds. Plant Disease, 69, 6–10. Tessmann, D. J., Charudattan, R., Kistler, H. C., & Rosskopf, E. N. (2001). A molecular characterization of Cercospora species pathogenic to water hyacinth and emendation of C. piaropi Tharp. Mycologia, 93, 323–334. doi:10.2307/3761654 Tessmann, D. J., Charudattan, R., & Preston, J. F. (2008). Variability in aggressiveness, cultural characteristics, cercosporin production and fatty acid profile of Cercospora piaropi, a biocontrol agent of water hyacinth. Plant Pathology, 57, 957–966. doi:10.1111/j.13653059.2008.01867.x Venter, N., Hill, M. P., Hutchinson, S.-L., & Ripley, B. S. (2013). Weevil borne microbes contribute as much to the reduction of photosynthesis in water hyacinth as does herbivory. Biological Control, 64, 138–142. doi:10.1016/j.biocontrol.2012.10.011 Walker, H. L. (1981). Factors affecting biological control of spurred anoda (Anoda cristata) with Alternaria macrospora. Weed Science, 29, 505–507. Retrieved from http://www.jstor. org/stable/4043339 Zhang, Z., Schwartz, S., Wagner, L., & Miller, W. (2000). A greedy algorithm for aligning DNA sequences. Journal of Computational Biology, 7, 203–214. doi:10.1089/106652700500 81478