ISSN No: Print: 2319 –1546; Online: 2319– 1554 Journal of Research in Ecology An International Scientific Research Journal Original Research Journal of Research in Ecology Pathogenicity of fungi associated with brown spot and leaf necrosis of Hydrangea macrophylla (big leaf Hydrangea) in Uyo, South-South, Nigeria Authors: Andrew DE1 and Akpan EA2 ABSTRACT: Disease i ide e su e s e e a ied out o o spot a d leaf e osis of Hydra gea acrophylla et ee Jul a d Septe e ; .The s pto s o se ed i the ield e e o spot hi h as i ula o slightl i egula i shape Institution: a d so e hat su ke o the lesh lea es of H. acrophylla. The e te of the spot 1. Department of Crop as a out i h i dia ete . S pto s o se ed i the ield i lude i ula o Science, Faculty of spot fou d p edo i atel at the ase of the leaf of H. acrophylla. The spot slo l Agriculture University of tu s ta to so e hat light g e e i led a pu ple halo. The spot as a out o e Uyo. eighth to o e fou th i h i dia ete . The i fe ted H. acrophylla sa ples e e 2. Department of Crop pla ed o the potato De t ose Aga PDA a d oist ha e . The esults sho ed Science, Faculty of that Colletotrichu gloeosporioides as the ost f e ue tl isolated o ga is o the Agriculture, Akwa Ibom i fe ted H. acrophylla lea es ith pe e tage f e ue ies of o u e e of . %, State University, Obio Akpa. hile Cercospora hydra gea had . % f e ue of o u e e. Pathoge i it o sus epi le H. acrophylla usi g all the fu gal isolates, sho ed that Corresponding author: C. gloeosporioides i ited o spot a d leaf e osis of H. acrophylla i this Andrew DE stud . The othe fu gi isolated also a ha e s e gized the disease de elop e t i this stud . Key ords: Hydra gea Pathoge i it test. acrophylla ig leaf Hydra gea , B o spot a d leaf e osis, Ari le Citaion: Andre DE and Akpan EA Pathoge i it of fu gi asso iated ith o spot a d leaf e osis of Hydra gea acrophylla ig leaf Hydra gea i U o, South-South, Nige ia Journal of Resear h in E ology : Dates: Re ei ed: Web Address: http://ecologyresearch.info/ documents/EC0526.pdf Journal of Research in Ecology An International Scientific Research Journal Fe A epted: Ma h Pu lished: Ap il This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. 1509-1515 | JRE | 2018 | Vol 6 | No 1 www.ecologyresearch.info Andrew and Akpan, 2018 INTORDUCTION Hydrangea (Reed, 1998; Reed and Rinehart, 2007). Wild plant is family Hydrangea used as tonic to the entire genito-urinary Hydrangeaceae. Hydrangea is a flowering shrub, easy system which includes the prostrate and also it generally to grow with different colours in the garden from mid- improves the health of the lungs, stomach and the pros- summer through fall (Fowler and Black, 2000). trate (Reed, 1999; 2000). It also cools the body and the Hydrangea begins flowering from early spring to late circulatory system (Pippa et al., 2000). H. macrophylla autumn. There are firmly packed in bunches towards the is affected by several fungal diseases which inflict end of the stems. They deliver vast sprouts that during heavy losses in its production. One of such fungal dis- late summer and autumn, they produce large blooms ease is the leaf spot disease. The field symptoms were with bold and beautiful colour. (Carter and Locke brown spot which was circular or slightly irregular in 1994). shape. The disease spread rapidly during frequent sum- belongs to the plant Hydrangea macrophylla is reported to have mer rains. The leaf spot disease showed significant im- originated in Japan. The name Hydrangea originates pact on the appearance, spectacular floral display and from the Greek word ("Hydro" which implies jar or market value of H. macrophylla. The disease attack also vessel') and is generally interpreted as "water barrel" reduces medicinal potential of the plant. This research which alludes to Hydrange’s requirement for a lot of work is to study the brown spot and leaf necrosis of water and its cup shaped flower (Reed, 2000). The col- H. macrophylla (big leaf Hydrangea) with the following our of Hydrangea could be blue, pink or purple and this objectives: is controlled by the acidity of the soil (Horst and Locke,  To isolate and identify organisms responsible for 1998). brown spot and leaf necrosis of Hydrangea This plant is a hard and tender shrub and some- times a woody climber. These are mostly deciduous macrophylla.  To determine pathogenicity test against microorgan- plants, though a few tender species are evergreen. isms. Hydrangea produces diverse flowers from the large globes of "mophead" to the discs of the "lacecaps", MATERIALS AND METHODS thick cones of oakleaf and panicle Hydrangea. (Reed et al., 2002).The height of Hydrangea ranges from dwarf (about2 - 3ft. high) to large bushes with stems of 9-10ft. (Reed, 1999; Reed, 2004). Hydrangea inclines toward well drained loamy and acidic soils for ideal p odu i it . It also thrives in the pH ranges of 4.5 – 6.5 enriched liberally with organic matter. Reed (2001). They requires very low temperature. (Jones and Reed, 2006) Hydrangea macrophylla is a medicinally important plant (Reed, 2004). Hydrangea root is used as syrup with honey and sugar or simply steeped in water and drank as tea. Hydrangea bark can be used for the Altogether two experiments were carried out, Table 1. Mean disease incidence and severity of the leaf spot disease of H. macrophylla S. No Days Sample plant Infected plants Disease incidence Disease severity 1 2 3 4 5 6 7 8 9 10 3 6 9 12 15 18 21 24 27 30 100 100 100 100 100 100 100 100 100 100 17 34 45 67 70 73 75 80 83 89 17 34 45 67 70 73 75 80 83 89 1 1 4 4 5 5 5 5 5 5 treatment of burns, bruises sparing and sore muscles Disease incidence: (%) Journal of Resear h in E ology : - Andrew and Akpan, 2018 Table 2. Fungi associated with brown spot and leaf necrosis of Hydrangea macrophylla and their percentage frequencies of occurrence S. No Fungi Number of pathogens Percentage frequencies of occurrence (%) 1 2 Colletotrichum gloeosporioides Cercospora hydrangea 9 1 90.0 10.0 one at shade house and one at the laboratory at the de- Collection and handling of samples partment of crop science, Faculty of Agriculture, Uni- The samples (H. macrophylla) were randomly versity of Uyo. Uyo is located in the tropical rainforest collected from D – line Ewet Housing Estate, Uyo, zone of Nigeria which is located in the 5º03’ N, 07º 56’ Akwa Ibom State. These samples were carefully collect- E ed (with clean surgical blades), placed in clean speci- and altitude 38m above mean sea level (Meteorological Garden, 2008). men bags and taken to the laboratory for investigation. Disease survey for the incidence and severity of leaf Isolation of associated fungi Infected H. macrophylla were washed in sterile spot disease of Hydrangea macrophylla Disease survey was conducted on the infected water to remove contaminant. The washed leaves were H. macrophylla field during the wet season (July to Sep- cut with sterile surgical blade into sections of 3-5cm tember) of 2009. A total of 100 plants were tested. The long, surface disinfected in 1% sodium hypochlorite disease incidence was acquired by counting the total solution for 10 minutes and was rinsed in the distilled number of diseased plants in the examined zone over water for several times. The cut leaf sections were plat- the total number of plants inspected. The disease inci- ed on the fresh Potato Dextrose Agar (PDA) and wet dence and severity rate and seriousness were recorded chamber. The dishes were incubated at 28+ 2ºc for five for 30 days Disease incidence and severity were as- (5-7) days. Successive sub-culturing of the isolates on sessed based on Wokocha (1990). fresh PDA medium was done to obtained pure cultures. The pure culture was stored as stock cultures on PDA slants inside McCartney bottles in the refrigerator at Disease incidence (I) = 150C until needed (Mbadianya et al., 2013). Disease severity was also recorded on the following Identification of pathogens Pure cultures were wet mount and observed scale: 0 = No disease observed under compound binocular microscope at 100 x and 40 1 = 14% of leaves is infected x amplifications for fungal growth. Based on their mor- 2 = 15- 36% of leaves is infected phological and cultural characteristics, fungal isolates 3 = 37- 50% of leaves is infected identified (Barnett and Hunter, 1987). The percentage 4 = 51 – 75% of leaves is infected frequencies of occurence of all the isolated fungi was 5 = 75 -100% of leaves infected. Wokocha (1990) calculated (Table 5). The presence of fungal growth was recorded each time (Wokocha and Aduo, 2011). The Table 3. Pathogenicity test of the isolated fungi S. No 1 2 Fungi Colletotrichum gloeosporioides Cereospora hydrangea Pathogenicity + D.I 84 D.S 5 0 0 - D.I: Disease incidence; D.S: Disease severity; +: pathogenic; -: Non- pathogenic Journal of Resear h in E ology : - Andrew and Akpan, 2018 H. macrophylla (Wokocha et al., 1986). Table 4. Disease incidence and severity of leaf spot disease of H. macrophylla caused by Colletotrichum gloeosporioides S. No Days 1 2 3 4 7 14 21 28 Disease incidence (%) 3 16 54 84 Characterization and identification of isolates The identification of isolated organism was Disease severity 1 1 4 5 done by comparing the characteristics of the culture with the characteristics of the known taxa. Barnett and Hunter (1987); Frank (2006). Study on the brown spot and leaf necrosis of H. macrophylla (fungal causative agent: Colletotrichum gloeosporioides). percentage frequency of occurrence of each isolate was After knowing the reason for the disease, we done as per Ebele (2011). Frequency of occurrence of different types of fungi= repeated shade house pathogenicity test with Colletotrichum gloeosporioides on H. macrophylla and disease incidence and severity was resolved from 100 Preparation of PDA plants. Data was collected for 30days after inoculation Potato Dextrose Agar (PDA) was prepared, au- and the symptoms were deliberately noticed. toclaved and poured into sterile petridishes. PDA utilized in this investigation was sourced from Center Biomedical Laboratories Ltd. Umoren Lane, Uyo (Mbadianya et al., 2013). RESULTS The mean disease incidence at 30 days and severity of the brown spot and leaf necrosis H. macrophylla. Preparation of inoculum suspension The isolates obtained were grown on potato dextrose agar. Hyphal mat from 5-7 day old PDA cultures of the pathogens were scraped aseptically on to a fine cheese cloth, filtered and washed in several changes of sterile distilled water to remove traces of stalling materials. The mat were then transferred aseptically into 200ml of distilled water solution in a warring blender and homogenized for one minute at a low speed in order to get the inoculate ready for pathogenicity test on Hydrangea macrophylla (Table 1) demonstrated that the rate of infection of the pathogen increased as sampling days expanded. The results demonstrated that for each 100 plant unit inspected, 17 plants were infected as at the 3rd day of the disease survey while 89% of plants were infected at the 30th day of the study. The disease severity (degree of damage) likewise follow similar pattern with the disease incidence. From the 18 th day to the 30th day, the severity was over 70% of infec- Table 5. The cultural, morphological and differential characteristics of fungal isolate S. No of Source of isolate Isolate Frank (2006) Barnett and Hunter (1987) 1 2 Colour of colony in mass Type of soma present Black Setae Black Setae Black Setae 3 Nature of the hyphae Aseptate Aseptate Aseptate 4 Special vegetative structure Broom like Broom like Broom like 5 Asexual spore (shape) Oblong Oblong Oblong 6 Special vegetative structure Erect wall Erect wall Erect wall 7 Conidia head Disc shape Disc shape Disc shape 8 Probable organism C. gloeosporioides C. gloeosporioides C. gloeosporioides Journal of Resear h in E ology : - Andrew and Akpan, 2018 tion. This results affirm that Hydrangea macrophylla is C. susceptible to fungal attack. C. gloeosporides had the highest percentage frequency Fungi associated with Brown spot and leaf necrosis of occurrence (90.0%) while C. hydrangea had the low- of H. macrophylla est frequency of occurrence (10.0%). C. gloeosporioides gloeosporioides and Cercospora hydrangea. A total of two (2) different fungi were isolated may be the primary inoculum causing primary infection. from infected H. macrophylla leaves (Table 2). They The results of the pathogenicity test of all the fungal were Colletotrichum gloeosporioides and Cercospora isolates showed that only C. gloeosporioides was patho- hydrangea. The isolated fungi were identified following genic on H. macrophylla. It produces the characteristics macroscopic and microscopic observation of their mor- symptoms of brown spot with alternating dark and phological characteristics and the help of identification slightly lighter rings of dead tissue which often give the guide (Barnett and Hunter, 1987), Frank (2006). spot a bull-eye target spot appearance which produce C. gloeosporioides has the highest percentage frequency the most severe effect at 28 days after inoculation. This of occurrence (90.0%) while Cercospora hydrangea had was similar to the characteristics symptoms described the lowest (10.0%) percentage frequencies of occur- by (Pirone, 1978). C. gloeosporioides was reisolated rence. from infected leaves of H. macrophylla. The observa- Pathogenicity test tion of the characteristic symptoms of brown spot with The pathogenicity test results of all the fungal alternating dark and slightly lighter rings on the leaves isolates (Table 3) demonstrated that only Colletotrichum and the reisolation of the pathogen from the infected gloeosporioides Hydrangea H. macrophylla showed that C. gloeosporioides incited macrophylla plants since it produces a replica of brown brown spot and leaf necrosis found on the leaves of spot which was circular or marginally irregular in shape H. macrophylla in this study. Other fungi isolated may and to some degree sunken on fleshy leaves of H. have been secondary invaders on the lesion caused by macrophylla. The re-appearance of these symptoms and the culprit pathogen and thus could not cause spot on reisolation of the pathogen from the infected H. the leaves. This observation is in agreement with the macrophylla plant showed that C. gloeosporioides is the report of Flentje (1965) and Wilhelm (1967) that such culprit pathogen in this study. Cercospora hydrangea organisms, when inoculated on the host in the absence also isolated may have played a synergistic role in the of the primary pathogen would have no effect. was pathogenic on disease development and progression (Table 4) shows the highest incidence (84%) and severity that were rec- REFERENCES orded at 28 days after inoculation. Ann Fowler and Timothy A Black. 2000. The plant of pennsylvania: an illustrated mannual, anna Anisko, il- DISCUSSION The result lustrator. Morris Arboretum, University of Pennsylvania of the disease survey on H. macrophylla taken for a period of 30 days indicated that the infection rate of the pathogen increased progressively as the sampling days increased. The degree of damage (severity) also increased remarkably press, 455-456 p. Barnett HL and Hunter BB. 1987. Illustrated genera of imperfect fungi. Burgress Publishing Company, Minneapolis. 241 p. in the same manner. Two different fungi were isolated Carter MR and Locke JC. 1994. Botanical oils alone from the infected H. macrophylla leaves. They were and combination with fungicides for the control of black Journal of Resear h in E ology : - Andrew and Akpan, 2018 spot (Abstr.). Phytopathology, 84:1077 ed. John Wiley and Sons, New York. 309-310 p. Ebele MI. 2011. Evaluation of aqueous plant extracts Reed SM and Rinehart T. 2007. Simple sequence used in the control of pawpaw fruit (Carica papaya L.) repeat marker analysis of genetic relationship within rot fungi. Journal of Applied Biosciences, 37: 2419- Hydrangea macrophylla. Journal of American Society 2424. for Horticultural Science, 132: 141-151 Flentje NT. 1965. Pathogenesis by soil fungi in: Ecolo- Reed SM, Joung Y and Roh MS. 2002. Inter specific gy of soil- borne plant pathogens (Baker KF and Snyder hybridization in clethra. Horticultural Science, 37(2): WC. Eds.) University of California press, Berkeley, 393-397. USA. 225-268 p. Reed SM. 1998. Prospect for hybridization of Frank MD. 2006. The identification of fungi: an illus- Hydrangea macrophylla and H. paniculata. Proc. trated introduction with keys, glossary, and guide to Southern literature. Conference, 324-326 p. St. Paul, Minn Publishers, American Phytopathological Society. 176 p. disease control. 16 ed. Ball publishing, Batavia. 97-107 p. of Hydrangea arborescence ‘Dardom’ x H. involucarta hybrids. Horticultural Science, 41(3): 564-566. Wokocha RC. 2013. Pathogenicity of fungi associated with the leaf spot disease of Eggplant (Solanum aethiopicum L.) in a derived savannah zone. Journal of Agriculture and Veterinary Science, 2(4): 51-57. Meteorological Garden. 2008. A publication of the department of geography and regional planning, University of Uyo, Akwa Ibom State, Nigeria. pollination procedure for flowering Dogwood. Journal of Environmental Horticulture, 17(2): 92 -94. Hydrangea macrophylla ‘Kardinal’ x H. paniculata. ‘Brussel Lace’ inter specific hybrids. Journal of Environmental Horticulture, 19: 85-88. Daniel Gilrein. 2000. American horticultural society diseases: the complete guide Journal of Environmental Horticulture, 18(1): 29-33. Reed SM. 2004. Self- incompatibility and time of stigma receptivity in two species of Hydrangea. Horticultural Science, 39(2): 312-315 Wilhelm S. 1967. Parasitism and pathogenesis of root disease fungi, in: plant pathology, problems and progress. 1908-1958 (C.S. Holton et al., eds) Central Book Depot, Allahabad. 356- 366 p. Pippa Greenwood, Andrew Halstead, Chase AR and and Research Reed SM. 2000. Compatibility studies in Hydrangea. Mbadianya JI, Echezona BC, Ugwuoke KI and pest SNA Reed SM. 2001. Verification and establishment of Jones KD and Reed SM. 2006. Production and verification Association, Reed SM. 1999. Development of labour efficient hand Horst RK and Locke JC. 1998. Soil treatment for th Nursery to preventing, identifying and treating plant problems. 1 st ed. Dorling Kindersley (DK) publishing, 196 p. Pirone PP. 1978. Disease and pest of ornamental. 5 Wokocha RC and Aduo BC. 2011. Pathogenicity of fungi associated with the cocoyam rot blight complex in South-Eastern Tropical Nigeria. Agriculture, Agro-Science Food, Journal Environment of and Extension, 10(3): 49-54 th Wokocha RC. 1990. Integrated control of Sclerotium Journal of Resear h in E ology : - Andrew and Akpan, 2018 rolfsii infection of tomato in the Nigeria savanna: effect of Trichoderma viride and some fungicids. Crop Protection, 9: 231-234 Wokocha RC, Ebenebe AC and Erinle ID. 1986. Biological control of basal stem rot disease of tomato caused by Corticium rolfsii (Sacc) Curzi in Northern Nigeria. Tropical Pest Management, 32(1): 35 – 39. Submit your articles online at ecologyresearch.info Advantages       Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@ecologyresearch.info www.ecologyresearch.info/Submit.php. Journal of Resear h in E ology : -