- ' WW" "" ‘mmmn THE EPIDEMIOLOGY. ETIOLOGY AND CHEMICAL CONTROL OF FUSICOCCUM AND PHOMOPSIS CANKERS 0F HIGHBUSH BLUEBERRY Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY PAUL EDMUND PARKER 1976 This is to certify that the thesis entitled THE EPIDEMIOLOGY, ETIOLOGY AND CHEMICAL CONTROL OF FUSICOCCUM AND PHOMOPSIS CANKERS OF HIGHBUSH BLUEBERRY presented by PAUL EDMUND PARKER has been accepted towards fulfillment of the requirements for Ph.D. degree in Plant Pathology ) - 4 / 9-1'L;(.,,'( L 1 ILL/C .',..’b("/'/_.—' Major professor Date 12/28/76 0-7639 ABSTRACT THE EPIDEMIOLOGY, ETIOLOGY AND CHEMICAL CONTROL OF FUSICOCCUM AND PHOMOPSIS CANKERS 0F HIGHBUSH BLUEBERRY By Paul Edmund Parker Michigan blueberry growers are faced with a serious stem canker problem. About 20% of Michigan's 9,700 acres of highbush blueberry are threatened by Fusicoccum canker and/or Phomopsis canker. Usually three or four stems are killed, but in severe cases the entire bush may be killed back to the soil line. With a perennial crop such a blueberry, a persistent canker problem can become an economically limiting factor in production. In the upper peninsula of Michigan, Fusicoccum canker is already the limiting factor in highbush blueberry production. Before the commencement of this research, little was known concerning periods of conidial release, periods of field infection, modes of infection or any good acceptable means of chemical control. The use of a Burkard Volumetric Recording spore trap allowed the surveillance of wind-borne conidia and ascospores in fields heavily infected by Fusicoccum canker. To monitor rain dispersal of conidia of Fusicoccum putrefaciens and Phomopsis Paul Edmund Parker vaccinii, a trapping device captured conidia dislodged and released by rain from stem canker pycnidia. Potted healthy highbush blue- berries placed beneath heavily infected bushes for one month periods yielded data concerning periods of natural field infection. Inocu- lations employing conidia and mycelial agar blocks placed onto wounded and non-wounded bushes showed whether wounding was necessary for infection to occur. To better define the infection process by E, putrefaciens, conidial inoculated epidermal sections were examined microscopically. The relationship between environmental stresses and infection by E, vaccinii of highbush blueberry was studied by inocu- lating drought- and freeze-stressed plants. Conidia of E, putrefaciens were not found in large numbers in the air spora. Wind dispersal does not appear to be of great importance in the infection cycle. Ascospores of Godronia cassandrae, the perfect state of_§. putrefaciens, were scarce even in areas where apothecia of 9, cassandrae were located in the lower peninsula of Michigan. Ascospores were entirely absent in the area sampled outside of the range of apothecia. Ascospores as agents in the organism's dispersal and subsequent host infection appear to be insignificant. Rain dispersal of conidia of both canker organisms com- mences with the onset of rain. The largest numbers of conidia usually occurred during late May rains. The numbers of conidia caught declined through the summer with no conidia of E, vaccinii being caught after the first week of September. A few conidia of Paul Edmund Parker 5, putrefaciens were observable well into the autumn. Periods of field infection were highest during late May for E, putrefaciens. This corresponded with the main periods of conidial release. Conidial inoculations of_[. putrefaciens to healthy susceptible plants gave the highest frequency of infection during this same period, mid to late May.' The mycelial inoculated (f, putrefaciens) wounded and non-wounded stems were consistently infected through- out the growing season. Healthy non-wounded plants exposed to natural field inocu- lum of P, vaccinii remained uninfected and healthy non-wounded plants inoculated under greenhouse conditions remained healthy. Wounds, due to mechanical or freeze-injury allowed P, vaccinii to infect and cause disease. Drought-stressed plants did not become infected. The use of Difolatan Flowable fungicide on a four or six week protectant spray schedule substantially reduced the number of cankers (up to a 95% reduction) due to E, putrefaciens. The reduction of cankers due to E, vaccinii was not as dramatic, due possibly, to a portion of the bushes being systemically infected beneath the soil surface. A program of protectant Difolatan sprays applied at regular intervals from spring through autumn should result in control of Fusicoccum canker. The response of Phomopsis canker to control would depend on whether the bushes were systemically infected or not. THE EPIDEMIOLOGY, ETIOLOGY AND CHEMICAL CONTROL OF FUSICOCCUM AND PHOMOPSIS CANKERS OF HIGHBUSH BLUEBERRY By Paul Edmund Parker A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1976 ACKNOWLEDGMENTS I wish to thank Dr. Donald C. Ramsdell for his suggestions, patience and understanding during the course of this study. His confidence in me and his willingness to help with the many phases of the research has made my graduate studies both fruitful and enjoyable. My thanks to Drs. I. Knobloch, J. Lockwood and J. Hart for their suggestions on various portions of this work and for serving on my graduate committee; to Mr. John Nelson of the Michigan Blueberry Growers Association, whose generous help and support made this research possible; and to the technicians and students within the department who have been encouraging and help- ful. I would also like to thank my wife, Eng, and my parents, Mr. & Mrs. Stanley F. Parker, for their encouragement and aid during the course of my schooling. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVIEW The Early History and Occurrence of Fusicoccum Canker The Genus Godronia . Peach Canker Caused by Fusicoccum amygdali Chemical Control of Fusicoccum Canker . History and Occurrence of Phomopsis Canker Chemical Control of Phomopsis Canker METHODS AND MATERIALS Field Locations for Fusicoccum Canker . Field Locations for Phomopsis Canker . . Trapping of Air Borne Spores of Fusicoccum Canker . Trapping of Rain Dispersed Conidia from Fusicoccum and Phomopsis Infected Bushes . . Periods of Field Infection for Fusicoccum. and Phomopsis Canker . . Conidial Inoculation Experiments with Fusicoccum putrefaciens and Phomopsis vaccinii Wound and Non- Wound Infection Studies of Fusicoccum and . Phomopsis Canker . . Phomopsis Canker, Systemic Infection Study . . Isolation from Leaves and Petioles for the Presence of F. putrefaciens and P. vaccinii . . Effect of Environmental Stresses on Phomopsis Infection : Infection by Ascospores of Godronia cassandrae . . Germination of Conidia of Fusicoccum putrefaciens and Phomopsis vaccinii at Selected Temperatures and Relative Humidities . . . Effect of Exogenous Nutrients on Germination of Fusicoccum putrefaciens Effect of Temperature on Mycelial Growth of Fusicoccum putrefaciens and Phomopsis vaccinii iii Page vi vii £0wa 09 21 22 23 TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVIEW The Early History and Occurrence of Fusicoccum Canker The Genus Godronia Peach Canker Caused by Fusicoccum amygdali Chemical Control of Fusicoccum Canker . History and Occurrence of Phomopsis Canker Chemical Control of Phomopsis Canker METHODS AND MATERIALS Field Locations for Fusicoccum Canker . Field Locations for Phomopsis Canker . . Trapping of Air Borne Spores of Fusicoccum Canker . Trapping of Rain Dispersed Conidia from Fusicoccum and Phomopsis Infected Bushes . . Periods of Field Infection for Fusicoccum and Phomopsis Canker . . Conidial Inoculation Experiments with Fusicoccum putrefaciens and Phomopsis vaccinii Wound and Non- Wound Infection Studies of Fusicoccum and . Phomopsis Canker . . Phomopsis Canker, Systemic Infection Study . . Isolation from Leaves and Petioles for the Presence of F. putrefaciens and P. vaccinii . Effect of Environmental Stresses on Phomopsis Infection : Infection by Ascospores of Godronia cassandrae . . Germination of Conidia of Fusicoccum putrefaciens and Phomopsis vaccinii at Selected Temperatures and Relative Humidities . . Effect of Exogenous Nutrients on Germination of Fusicoccum putrefaciens Effect of Temperature on Mycelial Growth of Fusicoccum putrefaciens and Phomopsis vaccinii Page vi vii LONG!» (A) 21 22 23 Page Histological Observations of Infection by Conidia of Fusicoccum putrefaciens . 23 Preliminary In Vitro Evaluation of Fungicides for Control of Fusicoccum and Phomopsis Canker . . . . . . . 24 Field Evaluations of Fungicides for I973 . . . . . . 25 Field Evaluations of Fungicides for l974 . . . . . . 26 RESULTS FOR FUSICOCCUM CANKER . . . . . . . . . . . 27 Trapping of Air Borne Spores . . . . . . . . . . 27 Trapping of Rain Dispersed Conidia . . . . . . . . 27 Periods of Field Infection . . 33 Conidial Spray Inoculations of Healthy Blueberry Bushes . 33 Inoculations of Wounded and Non- wounded Blueberry Bushes. 37 Isolations from Leaves and Petioles of Blueberry Bushes . 37 Infection by Ascospores of Godronia cassandrae . . . . 39 Effect of Temperature on Infection by F. putrefaciens . 39 Conidial Germination at Selected Temperatures and Relative Humidities . . . . . 39 Effect of Exogeneous Nutrients on Germination of Fusicoccum putrefaciens . . 39 Effect of Temperature on Mycelial Growth of Fusicoccum putrefaciens . 42 Histological Observations of Infection by Conidia of Fusicoccum putrefaciens . . . 42 Preliminary In Vitro Screening of Fungicides Active Against Fusicoccum putrefaciens . . . . 46 Field Evaluations for Chemical Control of Fusicoccum putrefaciens . . . . . . . . . . . . . . 46 RESULTS FOR PHOMOPSIS CANKER . . . . . . . . . . . . 50 Trapping of Rain Dispersed Conidia . . . . . . . . 50 Spore Trapping from Leaf Pycnidia . . . . . . . . 52 Periods of Field Infection . . 52 Conidial Inoculations of Healthy and Wounded Blueberry Bushes . . . . . . . . 52 Systemic Infection by Phomopsis vaccinii . . S4 Effects of Environmental Stresses on Phom0psis Infection. 54 Conidial Germination at Selected Temperatures and Relative Humidities . . . 56 Effect of Temperature on Mycelial Growth of Phomopsis vaccinii . . . 56 Preliminary In Vitro Screening for Fungicides Active Against Phomopsis vaccinii . . . . 56 Field Evaluations of Fungicides for Control of Phomopsis Canker . . . . . . . . . . . . . 60 iv Page DISCUSSION . . . . . . . . . . . . . . . . . . 62 Fusicoccum Canker . . . . . . . . . . . . . 62 Phomopsis Canker . . . . . . . . . . . . . . 65 LIST OF REFERENCES . . . . . . . . . . . . . . . 68 Table LIST OF TABLES Conidial Germination of Fusicoccum putrefaciens on Glass Slides in Glass Distilled Water at Various Temperatures . . . . Fungicide Control of Fusicoccum Canker of Highbush Blueberry (Earliblue cv.), W. Olive, Michigan, 1974 Fungicide Control of Fusicoccum canker of Highbush Blueberry (Jersey cv.), Ludington, Michigan, I974 . Systemic Infection of Earliblue cv. Highbush Blueberry by Phomopsis vaccinii . . . Conidial Germination of Phomopsis vaccinii on Glass Slides in Glass Distilled Water at Various Temperatures . Fungicide Control of Phomopsis Canker of Highbush Blueberry (Rubel cv.), West Olive, Michigan, 1974 . vi Page 40 48 49 55 57 GT Figure IO. ll. LIST OF FIGURES The Number of Air Borne Spores Collected in a Field Infected by Fusicoccum Canker - Ludington, Mich. 1974 The Number of Rain Dispersed Conidia of Fusicoccum putrefaciens Caught From Cankered Jersey cv. Highbush Blueberry Stems - Fruitport, Mich. l973 . The Number of Rain Dispersed Conidia of Fusicoccum putrefaciens Caught From Cankered Jersey cv. Highbush Blueberry Stems - Ludington, Mich. I974 . . . The Number of Rain Dispersed Conidia of Fusicoccum putrefaciens Caught From Cankered Jersey cv. Highbush Blueberry Stems - Ludington, Mich. l975 . The Number of Fusicoccum Cankers After Exposure of Potted Healthy Jersey cv. Blueberry Bushes to Natural Field Inoculum - Fruitport, Mich. l973 . . The Number of Fusicoccum Cankers After Exposure of Potted Healthy Jersey cv. Blueberry Bushes to Natural Field Inoculum - Ludington, Mich. 1974 . The Number of Fusicoccum Infections Resulting From Monthly Conidial Inoculations of Healthy Jersey cv. Highbush Blueberry Bushes - l973 . . . The Number of Fusicoccum Cankers Resulting From Monthly Conidial Inoculations of Potted Healthy Jersey cv. Highbush Blueberry Bushes - 1974 Effects of Exogeneous Nutrients on Germination of Fusicoccum putrefaciens conidia . Effect of Temperature on Mycelial Growth of Fusicoccum putrefaciens Cultured on PDA . Observations of Germination of Fusicoccum putrefaciens on Blueberry Epidermis . . . . . . . . vii Page 28 29 31 32 34 35 36 38 41 43 44 Figure 12. 13. I4. 15. 16. vaccinii Cultured on PDA . . . . . . . . . . 58 Page Effect of Various Fungicides on Mycelial Growth of Fusicoccum putrefaciens . . . . . . . . . . 47 The Number of Rain Dispersed Conidia of Phomopsis vaccinii Caught From Cankered Earliblue cv. Highbush Blueberry Stems - West Olive, Mich. 1974 . . . . 51 The Number of Rain Dispersed Conidia of Phomopsis vaccinii Caught From Cankered Earliblue cv. Highbush Blueberry Stems - West Olive, Mich. 1975 . . . . 53 HR Effect of Temperature on Mycelial Growth of Phomopsis Effect of Various Fungicides on Mycelial Growth of Phomopsis vaccinii . . . . . . . . . . . ,. 59 viii INTRODUCTION Fusicoccum putrefaciens Shear, and Phomopsis vaccinii n.f. nom., are the causal agents of two stem canker diseases of highbush blueberry, Vaccinium corymbosum L., in Michigan. Characteristic maroon brown bullseye cankers with darkly colored pycnidia are typical of Fusicoccum infections. Symptomatology for Phomopsis canker is not as distinct; often no clear defineable canker is visible. It is often difficult to differentiate between winter injury and Phomopsis infection. Branches appear dead with wilted, dried leaves remaining attached. Positive identification of Phomopsis canker requires isolation of the causal organism onto artificial growth media. Both fungi confront Michigan blueberry growers with serious economic loss. Northern areas appear to be predominantly infected with f, putrefaciens, central portions of the state have a mixture 'of E, putrefaciens and P, vaccinii, and in the more southern counties of Michigan and northern Indiana, 3, vaccinii is dominant (56). Bushes show the effect of cankers during mid-summer, when droughty conditions often exist. Death and flagging of individual branches are the most visible expression of disease. With [, putrefaciens, two or three stems are commonly infected in Michigan. Up to 40% of newly planted, two yr. old stock are killed in Nova Scotia. Older plants have up to 25% of their stems destroyed (ll). In Massachusetts, one to three branches are usually killed, though in some cases entire bushes may be killed back (66). Repeated infections by Phomopsis can kill mature bushes back to their crowns in Michigan. About 20% of Michigan's 9,700 acres of commercial highbush blueberry are either infected or threatened by the canker problem and it has been a limiting factor of produc- tion in the upper peninsula of Michigan (J. W. Nelson, personal com- munication) (35). The only recommended method of control has been field sanitation, whereby dead diseased branches are pruned out and burned. Due to the difficulty of locating all diseased branches, some of which may be deep within the thick crowns, sanitation has not been as successful as hoped, and both canker diseases have been increasing in importance. Data concerning spore liberation, natural infection periods and methods of good control have been incomplete, inconsistent or lacking. Such data for Phomopsis canker are completely absent from the literature. The aim of this research was to study the epidemiology. etiology and possible measures of control for both Fusicoccum and Phomopsis canker. LITERATURE REVIEW The Early History and Occurrence of'FUSicoccum Canker F) Godronia cassandrae f. sp. vaccinii Peck, the perfect state of Fusicoccum putrefaciens, was first noted and collected by Peck in 1885 from specimens of Chamaedaphne calyculata Moench (Andromeda calyculata, L.), Leather-Leaf, in New York (36). Shear in 1917 _ J (45) made the first collection of the imperfect state in association with a species of Vaccinium, from rotted berries of y, macrocarpon Ait, American cranberry. Collections of G, cassandrae from a Vaccinium species were made in Alaska in 1922 (7). Crowley in 1923 (12) made similar observations as had Shear with his observations of E, putrefaciens on cranberry in Pacific county, Washington. Stevens demonstrated E, putrefaciens as the common cause of end-rot diseaSe of stored cranberry and noted that the blossom end was the point of infection (51). Later, Stevens and Brain concluded that E, pgtrefaciens was the most important disease organism associated with end rot of cranberry in Massachusetts (52). Association of E, putrefaciens with blueberries was not reported until 1932, when Conners reported Fusicoccum canker of highbush blueberry in Canada (10). McKeen in 1958 was able to induce infection on blueberry plants inoculated in November and December with conidia of E, putrefaciens, but not on bushes which had been inoculated in June and July. McKeen felt that infection occurred sometime after June (33). In Nova Scotia, Creelman determined that the fungus overwintered as mycelium in cankers on living stems and in the crowns of infected bushes. Fusicoccum was able to maintain slow growth even at temperatures of 0°C. The role of ascospores in the infection process was in doubt, since Creelman failed to obtain infections (11). The first observation of Fusicoccum canker in F) Massachusetts was made by Zuckerman in 1958. Initial lesions I“; appeared on one-year old stems in late winter and early Spring. Sporulating pycnidia were abundant in eastern Massachusetts fields from March through mid July (65). In 1962, Barnes and Tweedie (1) observed symptoms of Fusicoccum canker on Earliblue, Jersey, Pemberton, Rubel and Stanley varieties of highbush blueberry in Chippewa county, Michigan. The main concentrations of infection were located in the eastern shore of Lake Michigan. A Hirst Volumetric Air trap operated from mid July through October showed conidia typical of E, putrefaciens present during the periods July 12-19, August 14-16 and from September 23-27, 1963. Weingartner obtained the fungus, E, putrefaciens, in culture from 40% of the isolations he made from diseased cankered blueberry stems collected throughout Michigan (58). He also observed conidia washing down stems during rainy periods from April through June. Godronia cassandrae apothecia matured during mid-July with discharge of ascospores occurring from August through September. Weingartner felt that ascospores were important in the disease cycle. His observations also showed that the perfect stage ocrurred with greater frequency in Michigan from Oceana county northward (55, 59). Infection of lowbush blueberry, Vaccinium angustifolium, Ait., has been observed by Weingartner and Lockhart (59, 31). Lockhart . observed Godronia cassandrae infected bushes on roadsides and improperly burned lowbush fields. Up to 5% of hedgerow bushes were infected with the canker. With a periodic three or four year burning cycle normally practiced with lowbush cultivation, infection due to E, cassandrae can be kept to a minimum (31). Weingartner suggested the possibility of wild lowbush stands as sources of infection for highbush fields in Michigan and he demonstrated pathogenicity of lowbush isolates to highbush cultivars (56). Experiments by Weingartner showed that isolates from wild Spiraea and Leather-Leaf growing in the vicinity of commercial fields were often infected with an isolate of E, cassandrae,which was also pathogenic to highbush blueberry (56). Lockhart performed inoculation tests on 12 different commercial varieties of highbush blueberry to determine their disease resistance of Fusicoccum canker. Results showed Jersey to be most susceptible, with 71% of the plants being infected. Earliblue, Johnson, Bluecrop, Coville, Berkeley, Pioneer and Blueray were moderately susceptible. Rancocas and Concord were reported by Lockhart to be resistant (29). Lockhart found that lesion develop- ment was greater on plants grown under cool temperatures (15°C days, 4.5 C°nights) than in warm growing conditions (21°C days and 9.5°C nights). Inactive lesions on plants grown under warm conditions became active on exposure to cooler temperatures. Shear had observed the ability of E, putrefaciens to grow at low temperatures (46). Weingartner felt that cool temperatures favored development of Fusicoccum canker (56). The only mention of leaf symptoms in blueberry on connec- tion with E, cassandrae was by Lockhart (30). Symptoms appeared as several spots or lesions per leaf showing up from July to September. Leaf spotting was confined to the lower branches of canker infected bushes. Lesion size ranged up to 10 mm diameter, with pycnidia occurring in the centers of the lesions. Varieties most susceptible to stem canker were also most susceptible to leaf spotting. The Genus Godronia The genus Godronia was erected by Mougeot and Leveille. It includes a "group of inoperculate Discomycetes with more or less urceolate apothecia and filiform to subclavate ascospores" (21). Groves has included the genus Scleroderris in Godronia, stating that there are no major differences between the two genera to warrant their separation. One difference is that Scleroderris has subclavate ascospores while Godronia has filiform ones.) The type specimen for Godronia cassandrae was collected from Leather-Leaf by Peck, and the pathogenicity of this isolate has never been tested against any species of Vaccinium according to Groves (21). Gremmen (20) suggested that E, cassandrae may not infect Vaccinium species. The possibility of various form species has been discussed by Groves and Smerlis (21, 48, 49). Groves (21) contended that it is impossible to differentiate "forms" on purely morphological features, though most isolates do appear to be restricted to a particular host genus. Smerlis, in experiments La. with the host ranges of g, cassandrae, has recognized a number of form species (49). Differentiation due to pathogenicity has resulted in the erection of three form species; f. betulicola, f. cassandrae and f. vaccinii. The status of f. allunae, f. spiraeicola and f. ribicola had not been experimentally looked at by Smerlis and their status was uncertain (50). FR 1 Weingartner's report of infectivity of isolates from Spiraea onto blueberry might tend to indicate that f. spiraeicola may better be placed in E, vaccinii (56). What does appear to be evident is A} that f. cassandrae is distinctly different in host range from f. vaccinii. While f. cassandrae infects members of Alnus, Betula, and Salix, f. vaccinii is limited to species of Vaccinium (50). Conidial relationships have been firmly established in six of the twenty-four species. According to Groves (21), Fusicoccum is unacceptable as a conidial state of Godronia. Fusicoccum was originally erected by Corda in 1829, based on an organism which has no similarities to the conidial states of Godronia. Topospora, the earliest available generic name for macroconidial stages of Godronia has been suggested by Groves as the proper name for macroconidial forms of Godronia. The imperfect state is still often referred to as Fusicoccum though Eglitis has referred to it as Topospora sp. (l6). Peach Canker Caused by Fusicoccum amygdalil A large amount of work has been done with the peach canker fungus, Fusicoccum amygdali Delacr. Groves questioned the validity of assigning both fungi, E, amygdali and E, putrefaciens, to the same genus, since conidia of the two organisms are not the same. Fusicoccum putrefaciens has slightly Curved, hyaline, 2-celled, fusoid conidia, while E, amygdali has hyaline, single celled fusoid conidia. Weingartner has made comparisons between the two in the mode of infection, but it could be dangerous to compare the biology of unrelated organisms. PR Inoculation studies with E, amygdali showed that infection I” in peach can occur at fresh leaf scars, bud scars, stipule scars, fruit scars and also through blossoms. 0n leaves and fruits, infec- tion occurred at points of wounding, worm holes and bacterial spot- 3 ting wounds. The main period of infection was thought to be from April through June. Guba (22) observed infection of current season's growth. Nodal and leaf axil areas were also considered important courts for infection (23). Nodal infection is via wounds with the main period of pycnidial development and subsequent infection being from September to October in Massachusetts (22, 23). Haenseler and Daines' earlier report mentioned the nodes as the main zone of infection. Erumpent pycnidia exuded spore tendrils during periods of moist weather (25). A delayed dormant application of monocalcium arsenite at '3 lbs/100 gal., oil at 1% qts./1OO gal.and Bordeaux mixture (4-6-100) resulted in reductions over a three year period of 96%, 94% and 30% respectively. Three applications each of Ferbam, Captan, and Phygon resulted in 25-35%, 48-62% and 60% reductions, respectively (25). Guba stated that Thiram, Phygon, Captan and lime sulfur were toxic to Fusicoccum (24). Chemical Control of Fusicoccum Canker For control of Fusicoccum canker on highbush blueberries, Creelman (11) tried four eradication treatments, lime sulfur 2% gal./ 100 gal., Dichlone 1 lb./lOO ga1., phenyl mercury acetate a pint/ 100 gal. and Dinitro ortho cresol (Elgetol) l ga1.lOO gal. Eradi- cation treatments were followed by four applications of Bordeaux .1 mixture (10-15-100), as a protective spray. There was no measure- oJ'_I_“" able reduction in canker numbers. -" W.)- Zuckerman (65) also failed to achieve any control using Ziram, Ferbam, or phenyl mercury lactate. Lockhart achieved up to I a 10 fold reduction in cankers on Bluecrop cv. using Erad (an organic mercury fungicide) at l pint/100 gal. Erad at 5 pints/ 100 gal., followed by Thiram, was less successful as a control. Murphy's canker paint (a mercury-based preparation), gave satis- factory results on Berkeley and Coville cultivars, but not on Bluecrop. Jersey bushes were injured by Erad at rates of 1 pint/ 100 gal. Conidia of cankers treated with Murphy's canker paint remained non-viable for twelve months (29). Control of Fusicoccum leaf spot on Jersey cv. was obtained using dormant mercury sprays (31). Scleroderris lagerbergii Gremmen (closely related to the genus Godronia) (21) canker on Pinus resinosa Ait., was controlled by applications of chlorothalonil (Bravo) applied seven times from late May to mid August. Difolatan 80% wettable powder gave reduc- tions of up to 67% (47). 10 Histogy_and Occurrence of Phomopsis Canker The earliest record of Phomopsis vaccinii was as an end and storage rot of stored cranberries in 1916 (43). Crowley (12) made collections of E, vaccinii from diseased berries of Vaccinium macrocarpon Ait., American cranberry, and Shear gE_§l, made similar EEK collections of the same fungus in 1924. Phomopsis vaccinii as a canker causal agent on E, corymbosum L. was first noted by Wilcox on samples from Massachusetts in 1934 (61). Isolations from infected twigs and root bark of young dis- eased seedlings showed the presence of E, vaccinii. Infected twigs were severely defoliated and young plants killed. In extensive experiments by Hilcox (62), blueberry bushes were spray inoculated with conidia of E, vaccinii and kept under bell jars to assure moist conditions for three days. Microscopic exami- nation of infected shoots showed 95% spore germination within 24 hours. Succulent shoots were killed within five weeks. Apparently the fungus entered at the point of new growth and proceeded down the stem. Woody stems inoculated in the same manner showed only small reddish colored lesions, but the fungus failed to colonize the stems. When leaves were inoculated, lesions quickly formed, coalesced and produced pycinidia. With the cultivars used by Wilcox, Katharine and Scammell, infection occurred in apparently healthy succulent shoots (62). Demaree and Wilcox postulated that E, vaccinii entered blueberries through leaf infections (15). 11 In all of the field observations made by Wilcox, the perfect state, Diaporthe vaCcinii Shear, was not observed. Perithecia were induced by growing the fungus on corn meal agar and exposing the plates to outdoor conditions from July through February. Black stromata developed perithecia of the Diaporthe-type scattered among pycnidia of E, vaccinii. Ascospores were two-celled, constricted ”El and biguttulate (63). Brown isolated E, vaccinii from highbush blueberry and con- sidered it as the causal agent of stem crown galling in Michigan, Massachusetts, New Jersey and Oregon (5). Weingartner was unable to U»* definitely associate blueberry galling in Michigan with a Phomopsis species (56). Zuckerman and Bailey reported a galling disease in 1956 which was similar in macroscopic symptomatology as that reported by Brown (64). In the literature concerning various diseases caused by Phomopsis species, a number of studies have indicated that wounding or weakening of the host plant predisposes it to infection by Phomopsis. Brown's work with the tumor disease of oak and hickory trees showed that the pathogen was apparently weak and entered by host through wounds (6). Wounding was required for infection of Asiatic chestnuts (2). Pirone reported that Gardenia cankers caused by Phomopsis sp. were initiated at the stem base where freshly cut surfaces furnished an excellent point for infection (38). In melanose of citrus, infection by Phomopsis citri occurs only in the natural openings which develop in the separation layer at the abscision zone (4, 18). Evidence indicates that drought stress may 12 predispose ornamental woody plants to Phomopsis infection (44). In 1961, Raniere attributed the sudden occUrrence of extensive death of blueberry stems by Phomopsis canker and Botryosphaeria canker to low temperature damage the previous winter which according to Raniere "may have predisposed many plants to invasion by various weak patho- gens normally unable to cause any measureable injury to vigorous plants" (40). In the case of Phomopsis blight of junipers (Phomopsis juniperovora), young non-wounded needles are susceptible to infection. Whether wounding or weak host plant conditions are required for Phomopsis sp., infections probably depends on the host plant (37). Chemical Control of Phomopsis Canker There has not been any chemical control reported for Phomopsis canker of blueberry. English achieved a 60% reduction in the incidence of cankers of Kadota fig trees when freshly pruned branches were covered with plastic caps. Phenyl mercuric acetate also resulted in a 60% reduction of branch infections. A mixture of Rhoplex AC-33 and Coromerc (a mercurial compound) painted onto pruned areas reduced infection of fig branches by 75% (17). Gill achieved satisfactory control of Phomopsis blight of junipers using Benomyl as a foliar spray at 2, 4 and 6 week intervals (19). Peterson and Hodges working with the same disease showed phenyl mercury fungicides useful when sprayed at weekly intervals. Benomyl and thiophanate methyl both controlled Phomopsis blight when applied at 14-day intervals from early June through late September (37). METHODS AND MATERIALS Field Locations for Fusicoccum Canker All field data for Fusicoccum canker were collected at the F. Schmucker farm in Fruitport, Michigan during 1973. The focus of canker infection was at the western edge of a field of Jersey cv. bushes. The area surrounding the field consisted of scattered patches of wild Spiraea, blackberry and bracken fern. Seven-day weather recording instruments, e.g., a hygrothermograph (Bendix Corp., Baltimore, Md.), and a recording rain gauge (Weather Measure Inc., Sacramento, Calif.) were set up in clearings between the blueberry bushes. A volumetric recording spore trap (Burkard Scientific Sales Ltd., Rickmansworth, Herts, England), and rain run-off spore trapping devices attached to cankered stems, were located in the same vicinity. Two-yr. old healthy Jersey cv. bushes were transplanted between diseased bushes for use in fungicide eval- uations. Potted healthy bushes were also employed as trap plants to determine periods of natural infection. Due to the potentially higher inoculum levels in more northern areas (57), field data for 1974 and 1975 were collected at the S. Sokolnicky farm in Ludington, Michigan. The field has had a long history of Fusicoccum canker, and the presence of Godronia cassandrae f. sp. vaccinii, the perfect state of Fusicoccum putrefaciens, had been reported by Weingartner (personal communication). Upon 13 l4 preliminary examination in the autumn of 1973, it was found that conidial inoculum was high, large numbers of Fusicoccum cankers were present and apothecia of E, cassandrae were present on debris beneath blueberry bushes and on dead stubs of older stems. Apothecia were not present at the Fruitport location. At Ludington, infection appeared to be most intense in the southern portion of the field where lowbush blueberry (Vaccinium aggustifolium) and bracken fern bordered it. Both devices for spore trapping were erected and humidity, temperature and rainfall were recorded with the instruments in the manner previously described. Leaf wetness duration was deter- mined by using a leafwetness recorder (M. De Wit, Hengelo, Holland). Potted trap plants placed among infected bushes served as indicators for periods of natural infection. A randomized complete block design (28) situated within the area of heavy infection was used for evalu- ation of foliar protectant fungicides. Field Locations for Phomopsis Canker Field observations for Phomopsis canker were made at the C. Petroelji farm in West Olive, Michigan for the 1973 growing season. The field planting consisted of Rubel cv. bushes naturally infected with Phomopsis vaccinii. Rain water run-off spore traps were attached to dead, apparently diseased blueberry branches. A hygrothermograph located between two bushes in the zone of infection recorded temperature and humidity levels throughout the duration of the field study. Two yr. old Berkeley cv. bushes were transplanted between diseased bushes for use in a protectant fungicide evaluation 15 experiment. Potted healthy trap plants were placed among diseased bushes to determine periods of natural infection as described for Fusicoccum canker. Field studies for 1974 and 1975 were made at the M. Wieppert farm in West Olive in which Phomopsis canker appeared to be more active. At this location bushes showed heavy flagging (stem wilt- ing); some were almost killed back to the crown by Phomopsis canker. Water run-off spore traps, a Bendix hygrothermograph and a Weather Measure recording rain guage were employed during 1974 and 1975. Potted trap plants were also used during both seasons. A randomized complete block design (28) was employed during both seasons to evaluate protectant foliar fungicide sprays. Trapping of Air Borne Spores of Fusicoccum Canker A Burkard Volumetric Recording Spore trap was used to sample the air spora for the presence of Fusicoccum pgtrefaciens conidia and ascospores of Godronia cassandrae during the growing seasons of 1973, 1974 and 1975. During 1973, the trap was located at the F. Schmucker blueberry field of Fruitport. Location of the trap was towards the northwest corner of the field where the numbers of cankers were high. In Ludington, the trap was situated in the southeast portion of the field, again where cankers were abundant. The trap was situated in a skip space between two bushes. The suction fan of the trap was powered by a 12 volt, D.C. motor with a 12 volt car battery as its power source. The suction rate of the trap was adjusted to 10 liters/min. The trap allows a continuous 16 record of spore catches for intervals of up to one week. Spore traps were changed weekly from late April through mid November. For spore counting, the tape was cut into lengths of 48 mm which is equivalent to 24 hrs. of spore trapping. Sections nicked in the upper left hand corner allowed proper orientation of the tape during counting. Cut sections were floated spore surface down in a petri dish of 1% aqueous Safranin O stain for five minutes to stain the spores. Following a rinse in distilled water to remove excess stain, tapes were placed on glass slides and allowed to dry. A counter-stain with Trypan Blue in 40% acetic acid was sometimes used to differentiate between fungal spores and other plant matter (3). Tapes were scanned using a Wild-Heerbrugg Microscope at 4OOX magnification with wide angle oculars. Counts were made on a daily basis. Trapping of Rain Dispersed Conidia from Fusicoccum and Phomopsis Infected Bushes Traps on seven different cankered stems were set up each season for each field study. Spore trapping was initiated in late April or early May and terminated in early or mid November. Plastic 7 cm diameter funnels were positioned around stems, beneath active Fusicoccum or Phomopsis cankers. A length of tygon tubing connected the funnel to a one gallon plastic milk container. Rain water passing over the pycnidia of the canker released and carried conidia into the funnels and collection vessels. Vessels were changed on a weekly basis. A 25 m1 aliquot from each trap was centrifuged at 12,000 RPM on a Lourdes Model AA-C clinical 17 centrifuge. One ml of distilled water was added to the resulting pellet of spores and plant debris. This was mixed on a Vortex mixer and the number of spores counted on a Levy-Hausser Hemacytometer. Collection of'conidia released from leaf pycnidia of E, vaccinii was accomplished in a similar manner. In late August when lesions appeared, leaves exhibiting actively growing pycnidia were placed into plastic funnels. New leaves were placed into the funnels weekly. Conidia counts were made the same as before. Periods of Field Infection for Fusicoccum and Phomopsis Canker Two yr. old potted Jersey or Berkeley cv. blueberry bushes were placed beneath heavily diseased bushes in the field to determine the periods of natural infection for Fusicoccum and Phomopsis canker. Plants were left for periods of one month from the end of April until the middle of November. After one month's exposure the bushes were returned to East Lansing and kept in isolation under field condi- tions. The following year the amount of infection which had occurred the previous year was assessed. Stem sections which appeared to be infected, and randomly selected healthy areas,were surfaced dis— infested with a 10% Chlorox (NaClO) solution and plated out onto PDA amended with 100 ppm Streptomycin Sulfate (PDA-S). The number of cankers per stem was recorded. Each positive diseased zone was counted as one canker. 18 Conidial Inoculation Experiments with .Eesagegsumseeteetesaeae_ene E! . . .. Conidia from monoconidial cultures of Fusicoccum putrefaciens and Phomopsis vaccinii were harvested by flooding petri dish cul- tures with sterile glass distilled water. The conidial suspension was passed through four layers of sterile cheese cloth to remove extraneous matter. The number of conidia was determined with a hemacytometer and their numbers adjusted to between 5 X 105 and 5 conidia/m1 with sterile glass distilled water. The conidial 1 X 10 suspension was then sprayed onto healthy two yr. old Jersey or Berkeley cv. bushes with a De Vilbis #15 atomizer powered by com- pressed air. Leaves and branches were thoroughly wetted before placing plants in a mist chamber for a period of 48 hrs., after which plants were removed and placed in isolation under field con- ditions. The amount of infection was determined the following year by isolating from suspected and apparently healthy stem sections onto PDA-S as previously described. Inoculations were made on a monthly basis from spring through fall. A total of five plants and one uninoculated control plant were used each month for each fungus pathogen. In order to test the effect of various temperatures upon infection by Fusicoccum putrefaciens, two yr. old potted Jersey cv. 5 bushes were sprayed with a spore suspension (1 X 10 conidia/ml) of E, putrefaciens. Placement of plants into premoistened plastic bags tied closed with string followed inoculation. Three different temperatures were maintained in growth chambers (Sherer Gillett Co.. 19 Marshall, Mich.) (10°C, 22°C, and 30°C) for a period of 48 hrs, to determine optimum temperature ranges for infection. Each temper- ature setting employed five inoculated plants and one non-inoculated control plant. After removing the plastic bag enclosures, plants were allowed to dry. Plants were kept under isolation in the field. Disease assessment was made in November of the same year. Wound and Non-Wound Infection Studies of Fusicoccum and Phomopsis Canker The necessity of host injury as a prerequisite for infection by E, putrefaciens and E, vaccinii was studied by inoculating wounded and non-wounded portions of 2 yr. old stems with blocks of mycelium from monoconidial PDA cultures of the appropriate fungus. Two yr. old Jersey or Berkeley cv. bushes were used as host plants for E; putrefaciens and E, vaccinii, respectively. Inoculation zones were swabbed with a 10% NaClO solution, then rinsed with sterile distilled water and wounded by puncturing the stem epidermis twenty five times in a 12 mm circular zone with a sterile dissecting needle. Inoculum was placed onto the disinfected zone, which was wrapped with sterile moistened cheese cloth followed by aluminum foil. Plants treated in the same manner except for the puncturing of the epidermis served as non-wounded treatments. Controls consisted of wounded and non-wounded plants inoculated with sterile blocks of PDA, treated and wrapped in the described manner. Following inocu- lation, plants were kept in a mist chamber for 48 hrs. Subsequently, plants were placed into isolation under field conditions. Evalua- tions of disease were made the following year in the manner 20 described in the preceding section. Two inoculations on each of 5 plants for each fungus were made each month. Wound and non-wound inoculations were made with both fungi in 1973. This was continued during 1974 with E, vaccinii, but only non-wounded inoculations were performed with E, putrefaciens during 1974. Phomopsis Canker, Systemic {‘3‘ Infection Study_ ' Bushes at the M. Wieppert farm in West Olive have been heavily infected with Phomopsis canker for the past three years. ' Pam Inna-.1 . ‘1 Determinations as to the extent of crown infection of severely diseased branches were performed by removing branches at crown level, surface disinfesting them and isolating from them onto PDA-S. Isolation from Leaves and Petioles for the Presence of E, putrefaciens and’E, vaccinii Leaves and petioles from bushes at the Fruitport and West Olive locations were randomly selected throughout the growing season, April through October. Isolations from surface disinfested sections of leaves and petioles were made onto PDA-S. Effect of Environmental Stresses on Phomopsis Infection Twenty 2 yr. old Berkeley cv. bushes were subjected to temperatures of -3°C for one hour. Plants were then inoculated with a E, vaccinii conidial suspension containing 1 X 105 conidia/ m1, followed by a 48 hr. period of light, constant mist. Two 21 groups of control plants, 20 per group, were subjected to either freezing with no subsequent conidial spray inoculation, or no exposure to the cold, but sprayed with conidia. Inoculations made during June 1975 were isolated from in November 1975 onto plates of PDA-S. Two yr. old Berkeley cv. bushes were drought stressed by depriving 20 bushes of water for a two week period during summer. After two weeks, bushes exhibited symptoms typical of drought stressed plants, i.e. wilting of leaves and succulent stems. Plants were inoculated and isolated from in the manner described above. Control plants consisted of drought-stressed, uninoculated and non-stressed inoculated plants. Infection by Ascospores of GOdFohia cassandrae Apothecia of E, cassandrae attached to dead blueberry branches were collected in Ludington and wired to wooden frames suspended over 2 yr. old Jersey cv. bushes in East Lansing. Groups of 5 bushes placed beneath the apothecia were removed to isolation following one month of exposure to the ascospore inoculum. New apothecia were added monthly. The following year isolations were made from healthy and possible infected stem areas. Germination of Conidia of Fusicoccum utrefaciens and Phomopsis vaccinii at Selected lemperatures and Relative HDmidities Freshly collected pycnidia of E, putrefaciens and E, vaccinii were placed into separate drops of sterile glass distilled I’HHJ'P‘JI LN . .—.—a—- i | 22 water and teased apart with a flamed dissection needle. The spore suspension was applied to slivers of glass cover slips, or into glass depression slides. The depression slides containing the spore suspension and additional glass distilled water were held in closed petri dishes to prevent excessive evaporation. Cover slip slivers with the dried conidial suspension were placed in a flask apparatus “3‘ consisting of a Gilson Differential Respirometer reaction vessel with a piece of floral clay in the center well to anchor the slivers. A saturated solution of K2504 effected a relative humidity of 98% when sealed with Parafilm (53). The substitution of water for the A“; K2504 allowed for conditions of 100% relative humidity. Groups of four flasks and four depression slides held at three different temperatures gave determinations for the amount of germination and germ tube elongation. One hundred conidia selected at random and counted for the number germinated gave the percent germination. Ten randomly selected conidia were measured with an ocular micro- meter to determine the average germ tube length. Effect of Exogenous Nutrients on Germination of FUsicoccum putrefaciens Freshly harvested conidia from cankers of E, putrefaciens were placed into either sterile glass distilled water, a sterile 1% solution of glucose or a sterile blueberry stem decoction. Preparation of the stem decoction consisted of boiling 100 grams of chopped blueberry stems in 400 m1 of glass distilled water for 8 hour, followed by autoclaving for 20 minutes. Spores were mixed 23 with each of the above solutions in glass depression slides and checked for the average percent germination and germ tube elonga- tion at eight hour intervals. Effect of Temperature on Mycelial Growth of Fusicoccum pptrefaCTens and‘ __PhomopSTs vaccinii Seven mm diameter discs of actively growing mycelium from monoconidial cultures of E. putrefaciens and E. vaccinii were placed in the center of petri dishes of PDA. The effect of four different temperatures (10°C, 15°C, 22°C and 30°C) upon radial growth was measured. Measurements of radial growth were recorded at eight hour intervals from groups of five plates at each temperature regime and expressed as sq. mm of fungal growth. Histological Observations of Infection by Conidia of Fusicoccum putrefac1ens In order to better understand the infection process of Fusicoccum canker, histological observations(rfgermination and infection on host stem surfaces were made. Infection chambers were constructed from plastic petri dishes with holes on the sides to allow stems to pass through, yet (maintain moist conditions. Freshly collected conidia were applied to stems with a pasteur pipette. The inoculated plant and its attached chamber was placed in a mist chamber for 48 hrs. Follow- ing the interval of mist, inoculated branch sections were removed, sectioned into 2 cm lengths and the epidermis stripped off. A 1:1 solution of 100% ethyl alcohol and concentrated acetic acid Ir- ~l 1 .‘. - 24 followed by lactophenol for 12 hrs., resulted in cleared epidermal tissue with a transparent consistency. Selective staining for conidia and elongating germ tubes was achieved with a 1% Trypan Blue stain-in 40% acetic acid (53). A staining time of 10-15 seconds followed by a rinse in lactophenol gave satisfactory stain- ing. Observations and Ektachome transparencies of strips mounted '3‘ in lactophenol on glass slides were made using a Wild-Heerbrugg I microscope with magnifications of 375x and 750x. Epidermal strips showing germinating conidia were trans- ferred from the glass microscope slide to an alcohol dehydration L-_ series. Tissue was started at 50% alcohol and run through 100% as described by Jensen (26). A 24 hour change of n-butyl alcohol and paraffin oil preceded paraffin infiltration. The embedded tissue was sectioned on an A0 Rotary "820" Microtome (American Optical Co., Buffalo, N.Y.). Sections were stained with either Safranin 0, counter stained with Fast Green or a combination of Phloxine and Fast Green. Preliminary In Vitro Evaluation of Fungjpides for Control of Fusicoccum and Phomopsis Canker Eight different fungicides were tested using poison agar, fungicide amended PDA. The fungicides and formulations tested were: Difolatan (Captafol) 4 lbs./ga1. flowable, Dithane M-45 (Maneb) 80% wettable powder, Captan 50% wettable powder, Tribasic copper sulfate 12.75% metallic copper, Benlate (Benomyl) 50% wettable powder, Cela W524 (Triforine) 20% emulsifiable concentrate, and 25 Sodium Pentachlorophenate 79% soluble powder. Varying concentra- tions of the fungicides on an active ingredient basis [0.1, 1.0, 10.0 and 100.0 pg/ml (ppm)] were mixed with sterile warm liquified PDA on a Vortex mixer. The fungicide amended agar plates were inoculated with seven mm discs of actively growing monoconidial cultures of the two fungi. Four plates per fungicide concentra- . tion, plus eight unamended control plates were employed. Heasure- ‘} ments of mycelial growth were made at twelve hour intervals to assess the amount of inhibition. 1' Field Evaluations of Fungicides for 1973 In an experiment at the C. Petroelje farm at West Olive, heavily Phomopsis cankered bushes were cut off by a rotary saw in an experiment by Dr. C. M. Hansen of the Dept. of Agricultural Engineering, Michigan State University, with the purpose in mind of rejuvinating the old bushes. The emerging growth was protected by two rates and two timings of Difolatan 4F at 2 and 4 qts./acre in 100 gal. of water and l and 2 lbs/acre of Benlate 50% wettable powder in 100 gal. water, applied at 3 and 6 week intervals using a power sprayer and a handgun. The plot was arranged in a random- ized complete block design using 4 bushes per replication and 5 replications per treatment. The amount of infection was determined the following year by counting the number of flagged (dead) stems per bush. 26 Field Evaluations of Fungicides for 1974 The experimental plot design for 1974 employed naturally infected field bushes to evaluate the effectiveness of Difolatan 4F in the control of Fusicoccum and Phomopsis canker. A randomized com- plete block design was used with each treatment replicated five times at Ludington and four times at West Olive. Applications were made of Emu"? three different rates of Difolatan 4F (2 qts., 4 qts., and 8 qts./ acre in 125 gal. of water/acre at the Ludington site and 62.5 gal. 2L“. , -. of water at West Olive) at two intervals (every four and six weeks). . Isl-(V 7.4 ~ Applications of fungicides commenced in late April at bud break and terminated in early November after leaf fall. One pint of spray mixture per bush and a pint per bush effectively covered leaves, stems and crown portions at the Ludington and West Olive sites, respectively. Evaluations of one year old stems the following year determined the amount of new infection. Due to the difficulty of positively recognizing Phomopsis cankers, flagged branches were equated as cankers. RESULTS FOR FUSICOCCUM CANKER Trapping of Air Borne Spores Counts of conidia were made from the spore trap tapes col- Fit. lected at the Fruitport location in 1973. Spores were present :- during April 24 to June 6; a total of 21 conidia were trapped. No conidia were observed on any spore trap tape after June 6. Spore | trapping was continued from April 24 through November 16, 1973. i No ascospores were observed at any time during the season. Sampling of the air spora in Ludington through the growing season of 1974 resulted in the capture of 79 conidia and 3 asco- spores (Fig. l). Nineteen conidia were recorded for May 4, the highest number for any one day. The first conidia were captured on April 29; the last date of capture was on May 23. During 1975, the Burkard spore trap was in continuous oper- ation from May 2 to November 7. Only three conidia were counted during May, with an additional two the first week of June. No other conidia or ascospores were trapped for the remainder of the $635011. Trapping of Rain Dispersed Conidia Conidia of E, putrefaciens were captured in rain water at Fruitport the first week spore trapping commenced, May 1-8, 1973 (Fig. 2). The highest number of conidia/m1 occurred during the 3 second week of trapping, May 8-15 when 55 X 10 conidia/ml were 27 28 WWVANIVH 5'". SBHONI 0 N6 ¢.o wd ad 0.. N; ¢._ m.. 3: ES 53 OMONON¢~NNONQQSN.O_mmvNOM 5 mm NN mm nm .N m. t n. n. : m h o n . mm (a I ss 0 O 4 . (a a . ¢ . o . I s a . — ¢ . . . . . .- . : . p .. . . 1 . a a a a .. .3388 . 52m .3... 25 En. 533 w. 0d v.5. £32 £20503 1.2.50 $303.3... 3 .3885 23... a s 3.02.00 .205» 2:00 :4 3 .3632 2.... .2... S o N 3110900qu HIVIOINOO H ( é '§)380dSOOSV 9 838041115 .40 UBBWON 29 a .n o... .832. 882.2 .2 .23 12 III 0 9 8 7 6 5 4 3 2 I. F 19.2 The Number of Rain Dispersed Conidia of Fusi- coccum putrefaciens Caught From Cankered Jersey cv. Highbush Blueberry Stems- Fruitport, M a M w :W m m n m m 9 m a ' d II P h. mm. MI! 0 9 .mal.‘ .w Mm 9.9 N be 3 u .uwl' "I‘ "“ " ""l “. wamaww | #0.: 3.32. .2352 4 8 2 2 I I 6 Ex 22:8 .oz mNIMNmmm mung mmw 0.1: mum Inn awn m mum-0N 034 mNIE 0:4 Sub 034 N 02415 1.2. _MI¢N 1:4... vNIQ 42. 910.42. 0.1m 151 m 437mm 22. film. 23... 919 231 910 237 n 22.. 1mm >52 mNINN ><2 NNum. >52 Elm >52 01. ><$ Date .12.7T T T 1.1 TO.208T .3 .5 B .4 1.5 OST 2.21.0 T T Rain Inches 3O caught. Conidial counts declined from June through August. The last countable number was present the week of August 14-28. Only trace numbers were noted beyond that date. Trace numbers continued to occur into late October. During 1974, the first conidia were not trapped until the second week of May (Fig. 3). Spore catches increased to a peak of 4 conidia/m1 the week of May 23-30. As in the previous 1.55 X 10 year, numbers of conidia fell off as the season progressed further; the last measureable number of conidia occurred the week of August 22-29. A few conidia were noted during periods in September through November. Conidia were first caught during the 1975 season from May 5-9, when 1.4 x 102 conidia/m1 were collected (Fig. 4). A slight increase occurred the week of May 16-23. The highest number 5 trapped for the entire season was 1.21 X 10 spores/ml during the period of May 23-30. The spore numbers fell off to lower levels of 1.59 x 103 to 2.2 x 103 during the month of June. For July through mid August they were still lower, ranging from 2.8 X 102 to 1.0 X 103/m1. Several rainless periods occurred during the growing season and resulted in no spores being trapped. Another peak catch of 3 conidia/ml conidia was noted the week of August 16-22 when 1.9 X 10 were counted. Numbers of conidia were sparse after the mid August peak with only trace amounts being caught after the week of September 26-October 4. Spore counting was continued until mid November. 31 llro.xvuoaa2._. 88353. .E .20.. N >421QN .54 O I 8 T 6 5 3 2 I d 1 NJ 1 4 q H d ‘ M q 1 d N M ‘1 d 1 q 1 i1 1 d w 4 7 1 u 9 . m . I. .. .I. V m c a 1 F u. M ._ Cl . 9 1 mm M . m d N Heaumium ea. .m n .m ENTQENW km. W Len M '1 38-8.83 «w d M L IIIIIIIII I mmiwwdad 2. u C P 1111111481963 .2. r m 1111111111 6.10.9.3 on O m . P o e 111111111111 $.63 2. ’ r .I m F 8 -11....031815... 0n n M v. u 11111111111 3-0.1.2 ~ .m N n %m 9-1.5.. :. 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L. .2... mm. «c u 1 __ 28-08.. mm. r e s v .2. -55.. 0 mm m. s I ----------- em e....-0~.§.. mm. mm M. m m ummt 111111. omega-fies. 00.. em a .u um 11 n. 52,-0.5... 2.. .h- a m m .mm-V 111111. 0.57% 8s. 00.. e .1. on 82-3 .8: cu. e mm. .1111. nu 8.2-0. .6: no. n ma III. 0. .8... 10 .8... so. 1 . . r p h p p . mm . .1! a .02-6 >02 mm. 4 2 O 8 6 4 2 O 8 6 4 2 E 3 2 2 2 I I I I I . M m“ I f0. 5 00000..» 020350". _E \ 22:00 .02 0 MW 33 Periods of Field Infection Trap plants placed in the field from April 24 to May 15, l973 failed to develop any infections due to f, putrefaciens (Fig. 5). Bushes exposed to inoculum during the exposure period, May lS-June l9, yielded 0.4 cankers per stem. Cankers continued to occur during the subsequent four monthly exposure periods, but in diminishing amounts. Plants exposed from October 27- November l7 failed to become infected. The highest numbers of cankers during 1974 were observed during the exposure period of from April 29-May 29 when 0.8 cankers per stem resulted (Fig. 6). There was a sharp decline to 0.l cankers per stem during June. The number remained from 0.l to 0.2 from June through August. Another peak of infection occurred dur- ing late August and September. Infection continued to occur from late September through late November. Conidial Spray Inoculations of Healthy Blueberry Bushes Plants inoculated with conidia in 1973 showed the highest amount of infection from the April 25 inoculation period, with 0.3 Cankers per stem (Fig. 7). The amount of infection declined on plants inoculated in May, June and July, and no infection resulted from the August inoculation. Infection levels increased again as a result of the September and October inoculations. In 1974 the early May inoculation did not cause infection. The first occurrence of f, putrefaciens infection and the highest amount of disease was evident on bushes inoculated during June, 34 3.. 00. 0.. r... hm a.» 0.~ A58... 5.... 1 6 O I a a m m. m. w. m 1.. h . 1.. 6 c. v | o 6 - . . m a w. m. p. m m. flag... 1. 1. 6 fl. .11. a m x 00.530 A. O :2 ~00. 304 . ..o .8. 8.1.8... . «.0 4 nd a .00 6003 =0... .nbm. £05 £323.... 105.000... 0.0.... .3302 0. 02.0.5 5.2.2.5 so :22. 2:00: 0080... .0 0.00090 .23 9.8.000 E03023... .0 .3602 2.... 0.0.... um .. was xsuoxuoo wnoooogsng 'oN 35 00.. n. . 00.0 00 ~ 2.0 No.0 .0.~ 02.0... 0.00 .I 6 1. 9 t m m n. m .0 . I . a. . m . 03. -28.. c. H m m N H. 1. N. 0.00.0 00.3.00 .4. Z Z Z Z I Z Z c. 1. 6 c. b 6 J1 1 1 4 d d d o N . .o a .. N 0 A n s 1 m. =0... .00... w 23 o. - 0.0 m =0... .00.. 0 atom - w u. m - 0.0 s v - M w -.- a . 0.0 m. c. 0.00.0 :00 . 2.0. 005.003.0031 . 0.. 50.000... 0.0.... .220... 0. 02.000 5.2.00.0 .00 00.00 2:00... 00:00 .0 0.000000 .23 0.3.000 0.0000200... .0 .0050... 0.... 0.0.... - 36 mum. I00=0.0000. .0 0.00 0. 0. 0. 0. . 00 .0 00 N 80 .80 000 .2. E... .0... .00 .o 0 .0 n s. m. ..0 m n a w Eco—n :am .. N.O m A. x. a .8. .8. .000 u. L 0 / m 0 R. W... 00-0. -. 000000 3.00030 000000.: .00 000.0... L . . 00:00... .0 003.2000... 3.0.000 30.00.... 0.0.... 0 0 N. 00.0000 002.020. 200000.00... .0 00.002 0.; .- 0... W. 37 when 0.4 cankers per stem were positively identified (Fig. 8). The July inoculation resulted in 0.22 cankers per stem. As the season progressed, resulting infection levels declined until no detectable cankers or infection resulted as a result of the October 8 inocu- lation. Inoculations of Wounded and Non-wounded Blueberry_Bushes Plants which had been wounded prior to inoculation with f, putrefaciens were consistently cankered, and infections resulted from all inoculation dates during l973. Areas surrounding the inoculation zones were often maroonish-brown in coloration with concentric bullseye configurations typical of Fusicoccum cankers. Sporulating pycnidia were sometimes observed towards the centers of typical cankers. E, putrefaciens was isolated from these cankers and other atypical areas showing possible infection. Non-wounded inoculated plants were also consistently infected at all dates of inoculation during l973 and 1974 with typical cankers and pycnidia being present in many cases. Other inoculated plants showed zones of discoloration from which E, putrefaciens was isolated. Uninoculated plants appeared to be free of cankers, and the pathogen could not be isolated. Isolationsfrom Leaves and Petioles ofrBluEberry Bushes Isolations made from leaves and petioles collected from April through October at the Fruitport location failed to yield f} putrefaciens. 38 '50... 20:44:00,: to PBS ex .. o\o_ ska nxo Oxh :xm mxm 1 l I .30. .. 8...... .5833 €3.35 .3 ~85. 2:8: 3:... .o 25:2: .3... .2250 :55! 69:. 23:33. $3.30 £38373 3 89.5... of. o .oE . _.0 N6 nd to 0.0 915' - maze/smquoo wnoooogsng °oN 39 Infection by Ascospores of Godronia cassandrae Bushes which were exposed beneath apothecia of 9, cassandrae were observed for infections the following year. Bushes appeared healthy in all respects. Isolations from randomly selected stem portions proved negative for the presence of f, putrefaciens. Effect of Temperature on Infection by E. putrefaciens Bushes exposed to a temperature of 10°C for 48 hrs. under moist conditions immediately following inoculation with conidia of E, putrefaciens. showed an average infection rating of 0.25 cankers per stem. At 22°C. 0.2 cankers per stem resulted. At 30°C there was no infection. Conidial Germination at Selected Temperatures and Relative Humidities There was no germination of E, putrefaciens conidia at either 98% of 100% relative humidity. Germination required free water. The best germination at the temperatures tested occurred at 2l°C and 30°C (Table l). Germination at 10°C was very low being 0.3%, and germ tubes were extremely short. Effect of Exogenous Nutrients on Germination of Fusicoccum putrefaciens The addition of 1% glucose or a blueberry stem decoction failed to significantly increase the percentage germination over that in sterile glass distilled water (Fig. 9). However addition of either 1% glucose or the stem decoction gave greater germ tube 40 .umcammms mcoz cowumuppawc\mmgoam cm» we mgumcmp was» scum mgh a .mmewu moss» umamuwpamc mm: acmsummgu sumo ”cowumompqmc\umucaou mng mmcoam umcucsg meow o.~_ m.mw m.ap ~.N¢ m.op ~.m~ -- m.o N.Ne o.o~ o.mp m.¢~ m.m m.- m.m o.¢ -- m.o -- m.o -- m.o -- m.o Aggy ARV Asnv A&v Aenv Aav Asnv “NV Aenv o.o «.0 o.o ”NV om Fm op u mcsuocmasmh ; oe ; mm g cm 5 mp aAanv spasm; mask acme new mfixv comumcmsgmw .mmczumgmasmh mzowgm> um Loam: umppwumwo mmmpu cw mmcwpm mmme :0 mcmwumwwguzm Esouoowmzm mo cowum=FEme pmwuwcou .p mam<~ 41 IOO b 90 ’ 80 P 70 b p 60 ' E 50» I. 40 B'ecocttlzns'fln 30 D——-D l' l 20 I. G ucoee | O as: Distilled a——o o‘ 3 I6 24 32 40 TIMEflinJ '50 BtueberryStem Decoction |30 . o——-a :1. choose “0 " 0———O GbuDistiM 90 ‘ Water A——A 0| 0 can» run: ELowsATIow (an) 8 3 8 F lg.9 Etteds of exogenous nutrients on I unionism conidia. sugn' tcmt dittetetence in °/. 8 I6 24 A L 324 TIME (hrs) ermiruotion of here was no germination between exogenous nutrient treatments and gloss distilled water. Exogenous sources of nutrients gave siqiticantly geoter meowes ot tion than glass distilled m. LS mtlbee 980.0L83 . 42 lengths than did the glass distilled water control (LSD P = 0.01 32). Effect of Temperature on Mycelial Growth Of FUsicoccum putrefaciens f, putrefaciens grew on PDA plates at temperatures of 10°C, 15°C and 22°C (Fig. 10), but no growth occurred at 0°C, 27°C and 32°C. 0f the temperatures tested, optimun growth occurred at 22°C, with maximum radial growth (the colony reached the edge of the Petri dish) occurring in 120 hrs. Cultures maintained at 10°C and 15°C grew more slowly (LSD P = 0.01 = 365). Histological Observations of Infection by Conidia of FuSicoccum putrefaciens At six and twelve hours following inoculation, conidia showed abundant germination and some germ tube elongation, but there was no visible evidence of penetration or infection by the fungus. Elongating germ tubes did not appear to orient themselves relative to the host stomatal openings (Fig. ll-A). After 24 hours under moist infection conditions, hyphal strands from the conidia were greatly elongated and in places of heavy concentration they formed a web-like network covering por- tions of the epidermal surface (Fig. ll-B). Within these extensive growths of mycelium appressorium-like structures were formed (Fig. ll-C). Sometimes isolated. germinated conidia showed round swellings at various points on their elongating hyphal strands (Fig. ll-D); these also had an appressorium-like appearance. 0n two occasions, elongating hyphae from f, putrefaciens conidia MYCELIAL GROWTH (sq. mm) 43 } ml :22: 1600» moor 1200» 1000» soo- soo» ' 400» 200+ ‘ - ' O 12 24 36 48 60 72 84 96 IOB IZO 132 TIME (hrs) Fig. 10 Effect of Temperature on Mycelial Growth of Fusicoccum putre- faciens Cultured on PDA. ""— Fig. 11 44 Observations of Germination of Fusicoccum putrefaciens on Blueberry Epidermis. A. Germinating conidium of f, putrefaciens with elongat- ing germ tube (arrow). Note that the orientation of the germ tube is not directed towards the host blueberry stem stomate. Web-like mycelium of f, putrefaciens on blueberry stem epidermis with numerous appresorium-like bodies (arrows). Enlargement of Fig. ll-B showing an appresorium-like body (arrow). Germinating conidium of f, putrefaciens with elongat- ing germ tube and an appresorium-like body at the distal end of the germ tube (arrow). A hyphal strand from a conidium of f, putrefaciens apparently entering the stomate of the host, highbush blueberry, stem (arrow). No appresorium-like bodies were associated with the apparent penetration. Conidium of F. putrefaciens on the epidermis of an inoculated highbush blueberry stem. 46 appeared to enter host stomata (Fig. 11-E) without formation of appressorium-like bodies. Sectioned tissue showed germinated conidia of E, putrefaciens on the cuticular surface of the stem epidermis (Fig. ll-F), but appressorium-like bodies, or other infection struc- tures were not observable. In cross sections of stomate and assoc- iated substomatal chambers no fungal tissue could be discerned. Preliminary In Vitro Screening of Fungicides ActiVe Against Fusicoccum putrefaciens Mycelial growth of E. putrefaciens was markedly inhibited by Difolatan. Bravo, Captan and Triforine. With Difolatan. growth was only measurable at 0.1 ppm (Fig. 12). The other fungicides tested failed to inhibit mycelial growth to the extent as did Difolatan. Field Evaluations for Chemical Control Of“FusiEoccum putrefaCTens In field evaluations at West Olive, Michigan. Difolatan 4F significantly reduced the numberiyfcankers by 52% to 65% as compared with the untreated control (LSD P = 0.01 = 0.21) (Table 2). There were no statistical differences between the various rates and timings of Difolatan that differed from the control highly significantly. The number of cankers on Difolatan treated bushes at Ludington (Table 3) were drastically reduced, with reductions ranging from 82% to 95% as compared to the control. There were no statistically significant differences between treatments (LSD P = 0.01 = 0.118). Amount at Growth (mm sq. Amount of Growth (mm sq.) Amount of Growth (mm sq.) Amount of Growth (mm sq.) c . l I I00 I0 I 0.l C Captan ppm 2000; l000» 0 l l l l 100 IO I 0.1 C Bravo ppm 2000» 1000» l I I G IOO 10 i 0.1 c Bordeaux ppm 2000 1000» L A L l c 100 I0 l 0.1 c Difolatan ppm 2000‘» I000» o . l 11 I00 lo I 0.1 C Tritorine ppm 2000» I000» 0 100 ID I 0.1 c Dithane lit-45 ppm 2000 I000» o L - I00 I0 I 0.l C Benlate ppm 2000- I000» oIlll I00 ID I OJ C Sodium pentochlorophenote PPM Fig I2 Ettect at Various Fungicides on Mycelial Growth at Fusicoccum putrefaciens. Measurements at Mycelial Growth Were Mode at 96 Hours Following Inoculation at Fungicide Amended PDA Plates CI Control. 48 TABLE 2. Fungicide Controla of Fusicoccum Canker of Highbush Blue- berry (Earliblue cv.), W. Olive, Michigan, 1974. - —— Rate per Acre of b c Application Mean No. Fusicoccum d Difolatan 4F Used ’ Interval Cankers/Stem on 6/20/75 2 qt. 4 weeks 0.19** 2 qt. 5 weeks o.24* ‘ 4 qt. 4 weeks O.18** 4 qt. 6 weeks 0.14** 8 qt. 4 weeks 0.l4** 8 qt. 6 weeks 0.l9** Untreated (Control) 0.40 aA randomized complete block design was used with 5 replications per treatment. bA knapsack sprayer was used for all spray applications. A total of 62.5 gal. water per acre was used per application. cTreatment periods during 1974 were made on the following schedules: Four week treatments were made on 4/25, 5/23. 6/20, 7/18, 8/15, 9/12, 10/11, and 11/18. Six week treatments were made on 4/25, 6/6, 7/18, 8/29, 10/11, and 11/22. dOne asterisk (*) indicates LSD (P = 0.05 = 0.16). Two asterisks (**) indicate LSD (P = 0.01 = 0.21). 49 TABLE 3. Fungicide Controlaof Fusicoccum Canker of Highbush Blue- berry (Jersey cv.). Ludington, Michigan, 1974 Rate per Acre of Application Mean No. Fusicoccum Difolatan 4F Usedb’c Interval Cankers/Stem on 6/20/75d 2 qt. 4 weeks 0.09* 2 qt. 6 weeks 0.21* 4 qt. 4 weeks 0.12* 4 qt. 6 weeks 0.08* 8 qt. 4 weeks 0.14* 8 qt. 6 weeks 0.06* Untreated (Control) 1.23 aA randomized complete block design was used with 5 replications per treatment. bA knapsack sprayer was used for all spray applications. A total of 125 gal. per acre was used per application. cTreatment periods during 1974 were made on the following schedules. Four week treatments were made on 5/2, 5/30, 6/27, 7/25, 8/29, 9/26, 10/12 and 11/22. Six week treatments were made on 5/2, 6/13, 7/25, 9/12, and 10/17. dOne asterisk (*) indicates LSD (P = 0.01 = 0.118). RESULTS FOR PHOMOPSIS CANKER Trapping_of Rain Dispersed Conidia No conidia of E, vaccinii were observed in any of the weekly collections made from late April through mid-November during 1973 at the C. Petroelje farm in West Olive, Michigan. Apparently, 2 either the branches selected were not infected with P, vaccinii or Sporulating pycnidia were not present on the diseased branches. The following two years, spores were trapped at the M. Wieppert farm in West Olive. Branches having actively Sporulating Phompsis pycnidia were selected. Conidial catches for 1974 ran from late April through mid September (Fig. 13). Few conidia were collected the first week. The number of trapped conidia increased to 44 X 103/ml of trapped rain water the week of May 23-30. Conidial numbers declined during June and July with none being caught during two rainy periods in late July and early August. A few conidia were caught the week of August 8-15. A trace number of conidia were trapped on August 29- September 5. which was the last occurrence of E. vaccinii spores for the season. In 1975 conidia were first noted from April 25-May 2, with 2 1.8 X 10 conidia/ml of trapped rain water. The highest numbers of 2 conidia were trapped the last week of May with 8.7 X 10 conidia/m1. Conidial numbers tended to decline beyond that date. No conidia 50 51 IL. It} ll.wo.x.ue&2._. gm .6 .20... 222IIINWMHMIWQCTC5QSZIO 1 1 4 A A W q 1 T 1 e d 41 d-J d 3 A 4 1 d Phomop- sis vaccinii Caught From Cankered Earliblue cv. Highbush Blueberry Stems - West Olive. Mich. 1974 Fig.13 The Number of Rain Dispersed Conidla at Q U I: E E E \ .2 .12 S U II .NIN. mum «Tn .mwm I I I .. nmwmlmu dad I I I I mNINN dad I . mm In. .034 IIIIIIII filo .gd IIIIIIIIIII I 1.634 ttttt . .wadIou :59 ..... nNIo.J:—. 0T0. .43.. 070 35.. IIIIIIIIIIIIIIIIIIIIII 0:55;“ .22. IIIIIIIII . I I I hNION .22. IIIIIIIIIIIIIII ONIQiE. IIIIIIIIIIIIII I . IIIIIIIIInToiR. . IIIIIII oiRFOm >43 IIIIIIIIII onInN i I I nNIo. >4! IIIIIIIIII me >4! H IIIIIIII I . mIN >4! MWIV IIIIIIII IIIIN>¢8I0~£K< m I I I u meo..¢a—( ~20 IBI- 16' 14’ 12 10 a» 6’ 41. DATE oz 52 were caught from June 27-July 11, a period of very low rainfall. Conidia were again present from July ll-August 22 with a large catch of spores occurring the week of August 16-22. No conidia were caught after the August 16 to August 22 trapping period (Fig. 14). even though a considerable amount of rain fell during several weeks after the last week of spore trapping. Spore Trapping from Leaf Pycnidia Spores were observed only during the week of August 16—22. 3 Two traps were employed; one yielded a total of 4.8 X 10 conidia/ 5 conidia per total 3 m1 of trapped rain water or a total of 4.22 X 10 volume of trapped water. The other contained 1.1 X 10 conidia/ml 5 of trapped water or a total of 7.54 X 10 conidia. No conidia were trapped beyond August 22 even though rainfall was abundant. Periods of Field Infection Trap plants which had been subjected to natural field inoculum of E, vaccinii either during 1973 or 1974 were examined the year following their exposure. No signs of Phomopsis canker were observed. Randomly selected areas from each branch were plated onto PDA-S, but no 2, vaccinii was isolated. Conidial Inoculations of Healthy and Wounded Blueberry Bushes All non-wounded Berkeley cv. bushes inoculated with conidial suspensions of P, vaccinii during the years 1973 and 1974 appeared healthy upon examination the year following their inoculation. 53 file £0. u. wanna... 3.0353. .2. .20... 0854208642086‘2 322222 lllll m <44 “a swan: umppwumwo mmm_o cw mmcwpm mmm—w co wvcwuum> mwmaoeoga we cowpm:.ELmu meuwcou .m m.m