69-20,954 WEINGARTNER, David P eter, 1939STUDIES OF CANKER AND STEM BLIGHT DISEASES OF HIGHBUSH BLUEBERRY fVACCINIUM CORYMBOSUM L.) IN MICHIGAN. Michigan State University, Ph.D., 1969 Agriculture, plant pathology University Microfilms. Inc., Ann Arbor, Michigan STUDIES OP CANKER AND STEM BLIGHT DISEASES OP HIGHBUSH BLUEBERRY (VACCINIUM CORYMBOSUM L.) IN MICHIGAN fey David Peter Weingartner A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Botany and Plant Pathology 1969 ABSTRACT STUDIES OP CANKER AND STEM BLIGHT DISEASES OP HIGHBUSH BLUEBERRY (VACCINIUM CORYMBOSUM L . ) IN MICHIGAN by David Peter Weingartner The initial objectives of this research were to determine primary causes of cankers and s t e m blights of highbush blueberries in Michigan. Several fungi known to cause cankers and/or stem blight of highbush blueberries were associated with dying stems in Michigan blueberry fields. In addition, various types of overgrowthB reported to be caused b y other organisms were associated with some cankers. Application of K o c h ’s Postulates showed that G o d - ronia cassandrae Peck f . vaccinii Groves and Diaporthe (Phomopsia) vaccinii Shear caused cankers and stem blights of blueberries in Michigan. The diseases were renamed God- ronia (F usi coc cum ) canker and stem blight and Phomopais canker and stem blight, respectively. Red-brown to maroon elliptically shaped lesions, often centered by a leaf scar, were usually diagnostic for Godronia infection on 1- and 2-year-old stems. One- a nd 2- year-old stems which were blighted, but not cankered, we re usually infected with Phomopsis. On older stems oankers caused by G odr oni a tended to be short and wide whereas Phomopais cankers were often long, narrow and covered w i t h David Peter Weingartner unbroken bark. Since both fungi caused canker and stem blight symptoms, isolations were often necessary to confirm diagnoses made in the field. G o d r o n i a t and to a lesser degree, Phomopsis. were associated with certain types of calluses occurring along cankers on affected blueberry stems. Although calluses did not devel op on plants artificially inoculated with Godronia or Phomopsis. calluses similar to those observed in the field were Induced by mechanically girdling actively growing stems Some calluses may result from the slow girdling action of cankers caused by these fungi. B o t h diseases were widely distributed in Michigan, but G bdronia (Fusicoccum) canker and stem blight was con­ sidered more important because, in the field, major varieties of blueberry grown in Michigan were more susceptible to this disease. Blight symptoms caused by Godronia were more severe when artificially inoculated plants were grown under condi­ tions inducing dormancy of the host. The disease cycle of Godronia (Fusico ccu m) canker and stem blight was studied. That isolates of G. cassandrae from Sp i r a e a s p p . and V. angustifolium were pathogenic on Jersey variety blueberries suggested that these hosts may serve as inoculum reservoirs for the disease. Two major infection periods, 29 May - 10 July and 21 Aug ust - 9 October, were identified whe n different sets David Peter Weingartner of healthy 1 - and 2 -year-old plants were exposed to natural inoculum during the 1968 growing season. Conidia were moat abundant and were observed washing down stems during AprilJune whereas ascospores were discharged during AugustSe pte m b e r . Over of all lesions on stems collected in April occurred at leaf scars. Isolations from attached petioles collected from the field in October showed that \\.$% were infected by Godronia. When plants were spray inoculated 2-3 weeks before leaf drop, 53 of 66 lesions occurred at leaf scars. Attached petioles inoculated 0, 1, 2, and 5 days after removing leaf blades resulted in 3 k t 6 # leaf scar infections, respectively. scars on dormant plants were inoculated, 3 1 * 8 * and When healed leaf lesions did not develop until 1 year after inoculation whereas lesions developed immediately following inoculation when leaf scars were wounded before inoculation. It was concluded that leaf scars are probably infected via attached petioles before leaf drop. Infections produced by inoculating nonwounded stem internodes and histological observations of incipient necrosis below stomates suggested that stomates also ser­ ved as infection courts on 1 - and 2 -year-old stems. Histology of developing cankers showed that necro­ sis first appeared below stomates. Godronia initially grew through longitudinal air channels in the living stem cortex. David Peter Weingartner Hyphae were observed in Advance of necrosis, but chloroplasts of cortex parench^;ma turned red in advance of hyphae. cells. Hyphae grew alon g, but did not penetrate living Vessels in disco ored xylem were occluded with hyphae, various deposits and possibly tyloses suggesting that steins wilt because of vascular occlusion. DEDICATION To Sharon and Kris ii ACKNOWLEDGMENTS I want to acknowledge Dr. E. H. Barnes who started me on this research and helped in many ways until his untimely death in November, 1967? and Dr. E. J. Klos who has directed m y research and has unselfishly provided assistance since Dr. Barnes' death. I want to thank each member of my graduate commit­ tee, D r s . E. S. Beneke, W. J. Hooker, and J. E. Moulton for their advice, use of facilities and evaluation of the m a n u ­ script. Without funds and plant material supplied by the Michigan Blueberry Growers Association, not have been possible. this research would Thanks also to Mr. J. W. Nelson, Research Director for the Association, who helped in many ways. Thanks are due to Dr. W. G. Fields who helped identify some fungi and who often just listened.... I want to express appreciation to Drs. E. Smerlis, A. W. Stretch, R. J. Friend, B. M. Zuckerman, and C. L. Lockhart f o r providing cultures of G. caasandrae. Photographic credits are due to the following. Mr. Philip Coleman: Figs. 1-C, D; 2-C, D; 3-B, C, D; ij.-A, C; 5-A; 6 -C; 7-A to I; 8 -Aj 10-D; 11-A, D; 1£-B; iii 16-A, B, iv C. Michigan State University Photographic Laboratory: Pigs. 1-B; 2-A; 5-B, D; 8 -B; 1£-A, D. Pigs. £-E; 6 -B; 12-Aj and 31* Mr. J. W. Nelson: Barnes: Dr. P. H. Wooley: Dr. J. E. Moulton: Pigs. 2-B; 3-A; I4.—B ; 5-C. Pig. l^-E. Pig. 1L|.. Dr. E. H. Canadian Department of Agriculture: Pig. Ij.-E. Finally, X express my sincere gratitude to my wife Sharon and daughter Kristin for making the many sacrifices pursuant to my studies. TABLE OP CONTENTS Page D E D I C A T I O N ................................................. ii ACKNOWLEDGMENTS........................................... ill LIST OP T A B L E S ........................................... vii LIST OP F I G U R E S ............................................. x INTRODUCTION ............................................. 1 LITERATURE R E V I E W ........................................... 3 Introduction ........................................ 3 Stem blight and die-back diseases of blueberries . 3 Canker diseases of blueberries . . . ............... 6 Overgrowth, gall, and tumor diseases of blue­ b e r r i e s ............................................ 16 PART I. SYMPTOMATOLOGY................................... 20 Methods and M a t e ria ls ................................. 20 Association of organisms with symptoms . . . . 20 Isolation of organisms from diseased tissue. . 22 I n o c u l a t i o n s ............... 22 Experimental Results ............................... 37 Association of organisms with symptoms . . . . 37 Inoculation of plants in the greenhouse. . . . lj.2 Field s t u d i e s ......................................56 Studies of naturally infected plants ......... $ 6 Observations of plants tagged in UMF and NLM f i e l d s ............................. 57 Observations of plants tagged in CLM field . . 60 Survey of wilted branches........................ 62 Inoculation of plants in the f i e l d ............. 80 Low incidence of wilt on inoculated plants . . 84 Effect of dormancy on localized lesions. . . . 87 Effect of temperature on lesion development. . 88 Callused cankers ............................... 92 v Page PART II. DISEASE CYCLE AND INFECTION STUDIES. ... I n t r o d u c t i o n ............... Methods and M a t e r i a l s .................... .............. Availability of inoculum Other inoculum sources ........... . . . . . Infection p e r i o d s ......................... 100 Experimental Results ............................. Availability of i n o c u l u m ................ 102 Other inoculum sources .................. Infection p er i o d s ......................... 115 Infection sites. . . . . . . . . . . . . . . Infection of leaf scars prior to leaf drop . Infection of healing leaf s car s ............... Infection of healed leaf s c a r s ............... Infection of in ter nod es ........................ PART III. 99 99 99 99 99 102 102 121 121}. 12k 126 126 PATHOLOGICAL H I S T O L O T Y ................ 126 Methods and M a t e r i a l s ......................... 128 Experimental Results ............................. D I S C U S S I O N ......................................... 139 LITERATURE C I T E D ...................................152 vi 136 LIST OP TABLES Table Page 1. Godronia caaaandrae as interpreted by t J. W. Groves....................................... 6 2. Susceptibility of varieties of blueberry to infection by Godronia caaaandrae f . vaccinii.......................................... llj. 3* Ingredients and abbreviations of media used in this research................................. 21 ii.* Fungi isolated from stem sections taken from cankered and/or blighted blueberry plants . i 38 £. Sporulating fungi identified on sections of diseased blueberry stems placed in moisture chambers ............................. 6. Association canker fungi with symptoms observed on blueberry s t e m s ............. 39 . . I4.O 7. Results of artificial inoculations of blueberry plants with isolates of Go dronia, Pho mopais. and C o r y n e u m ............. 1+2 8. Fungi associated with symptoms resembling cold injury, Phomopsis, and Botrytis twig b l i g h t s .................... '.................... 61 9. Characteristics of leaves on branches killed by Godronia or Phomopsis .............. 72 10. Average ages of branches killed by Godronia and P h o m o p s i s ................................... 72 11. Association of Godronia and Phomopsis with symptoms observed on wilted stems . ......... 73 12. Association of Godronia and Phomopsis with symptoms on sections from stems collected in fields in which either Godronia (GDFields) or Phomopsis (FH-Fields) was the only pathogen isolated.......................... 76 13* Association of Phomopsis and Godronia with symptoms observed on sections of stems with discolored xylem ......................... vii 78 Page Table 11*.. 15. Association of Godronia and Phomopsis with varieties of blueberries grown in Michigan and Indiana. 81 Association of Godronia and Phomopsis with varieties of blueberries grown In Michigan 82 16 . Relative susceptibility of major Michigan blueberry varieties to Godronia and Phomopsis.................. .............. 83 Results of inoculations made on Jersey variety plants in the field with G o d ­ ronia and Phomopsis. ................. 85 Expansion of lesions and formation of pycnidia on plants inoculated with Godronia and grown at 5, 15, and 18 C. . 90 Expansion of lesions on plants inoculated with Godronia and grown for 10 days in a high humidity chamber at 18 C and then for 23 days at 5 and 15 C ............... 90 20 . Organisms associated with callused cankers 96 21. Presence of Godronia spores in fruiting bodies at v a r i o u s t i m e s during the 19651968 growing seasons .................... 103 Growth of forms of G. caaaandrae at various temperatures ............ ............... 105 23. Spore morphology of forms of G. caaaandrae found in Michigan........................ 106 2J+. Pathogenicity of forms of G. cassandrae on Jersey variety blueberries. . . . , 116 Godronia infection sites on 1- and 2-year old stems collected in April, 1968 . . . 122 Percent total infection, nodal infection, and internodal infection by Godronia occurring as lesions of various sizes. . 122 Percent Godronia lesions of various sizes occurring at nodes and internodes• • » • 122 Infection of necrotic leaves, petioles, buds and stem internodes by G. cassandrae 123 17. 18. 19. 22. 5. 2 26. 27. 28. viii Table Page 29• Infection of leaf scars by Godronia when suspensions of conidia were sprayed on plants 2-3 weeks before leaf d r o p ............125 30. Infection of leaf scars via petioles by Godronia at various times after removing leaf blades........................... .. ix 125 LIST OP FIGURES Figure 1. 2. 3- Page Lesion-young canker infection type on 1and 2-year-old s t e i n s ........... . ............. 26 A. Incipient lesions (red spots) on 5“ month-old stem collected from an upper Michigan field 3 December, 1968. B. Lesions of various sizes on a 1-yearold stem collected from an upper Michigan field 15 June, 1 9 6 7 . C. Lesion at the base of a Ij.- to S - y B & v old stem collected from a northern lower Michigan field 15 October, 1965* D. Typical lesions on 1-year-old stems collected in midsummer. Lesion-young canker infection type on 1- and .2-year-old stems................................. 28 A. Lesion on a 1-year-old stem collected from an upper Michigan field 15 June, 1967- B. Typical "bullseye" lesion with pycnidia on a stem collected in late May. C. Young canker on a 1-year-old stem col­ lected in July. D. Young canker on a 2-year-old stem col­ lected 15 October, 1 9 6 5 . Developed canker infection type on older stems. 30 A. Canker and depression on 3 “ to l4.-yearold stems. B. Developed canker on a l^-year-old stem. C. Mild expression of callus type 8-E. D. Callus type 8-E. x Page Figure lj.. Callused. blueberry steins.......................... 32 A. Callus type 8-A on a 3-y©ar-old stem of a Stanley variety plant. B. Callus type 8-A on Pemberton variety. C. Callus type 8-B on Earliblue variety. D. Callus type 8-B on Earliblue variety. E. Photograph of crown gall on blueberry provided by the Canadian Department of Agriculture. Callused blueberry s t e m s .................. .............. 6. 7* A. Callus type 8-C on Jersey variety. B. Callus type 8-C. C. Crown gall (?) symptoms on blueberry in Michigan. D. Callus type 8-D. E. Callus type 8-D. Miscellaneous symptoms associated with blight and canker diseases of blueberry in Michigan. A. Discolored bark or epidermis infection type on an unnamed variety photographed in May. B. Split or flaking bark infection type on 3-year-old stem photographed in late June. C. Split or flaking bark or epidermis infec­ tion type on 2- to 3-year-old stems. D. Discolored bark or epidermis infection type on 1-year-old stem of an unnamed variety photographed 29 May, 1968. E. Discolored bark or epidermis on a 2year-old stem photographed in May. Inoculation results ............................. A. Wrapped inoculation site. B. Control inoculation. C. Response on Jersey variety 10 days after inoculating with G. cassandrae f. vaccinii. xi 3k 36 k$ Page Figure 7. 9. 10. (continued) D. Response on Jersey variety 9 weeks after inoculating with G. cassandrae f. vaccinii. E. Canker on Jersey variety 11 months after inoculating with G. cassandrae f . vaccinii. F. Response on Bluecrop variety 10 days after inoculating with £. v a c c i n i i . G. Lesion on Bluecrop variety 10 days after inoculating w i t h P. v a c c i n i i . H. Young shoots which wilted 10 days after inoculating with J?. v a c c i n i i . I. Jersey variety stem wilted 6 weeks after inoculating with G. cassandrae f . vaccinii. A. Typical plant inoculated in studies performed in the greenhouse. B. Stem naturally infected (a) with Godronia and artificially inoculated stem (b). C. Section through acervulus of m i c r o s tictum on a blueberry stem (I4.OO X) . Percent maximum growth of Phomopsis vaccinii and Godronia cassandrae f. vaccTrTii at various temperatures. T . .................... J4.8 Naturally occurring and artificially induced symptoms on blueberry stems .................. A. Callus formation f r o m strip of cambium left protruding into mechanically g i r ­ dled portion of a 1-year-old stem. B. Naturally occurring callus along edges of a canker caused by G o d r o n i a . C. Gray centered lesion and m aroon colored epidermis after a plant was inoculated with Godronia and grown for the summer and following winter in a cold frame* xii $1 Page Figure 10. (continued) D. 11. Large canker on the crown of a 3-ye& r ,_ old plant inoculated with Godronia via a chip wound. Results of artificial inoculations............... £3 A. Lesion on Jersey variety plant 11 weeks after inoculation with G. cassandrae f . spiraeicola. B. Control plant (a) and plant ino cu­ lated with P. vaccinii (b) several months after inoculation. C. Canker on 2-year-old Jersey variety stem 6 months after inoculating with vaccinii in the field during May. D. Lesion on stem of Jersey variety plant-11 weeks after inoculating with G. cassandrae f . vaccinii from V. angustlfolium" E. Lesion on Bluecrop variety plant 6 months after inoculating with vaccinii in the greenhouse. . * 12. 13• Plants artificially and naturally infected wi th £. v a c c i n i i ...................................... 5>5 A. Natural infection on a i|.-year-old stem of Weymouth variety. B. Long, narrow canker on l4.-year-old Jersey variety stem inoculated in the field wit h £. va cc ini i. C. Sharp demarcation between living and dead tissues on a 1-year-old stem inoculated with £. vaccinii in the greenhouse. D. Pycnidia in necrotic tissues on a 1-yearold stem inoculated with jP. vaccinii in the greenhouse. Artificially induced Godronia l e s i o n s ...........6l|. A. Lesions which developed on 6-ruonth-old stem which was kept in a cold room for 6 months. xiii Page Figure 13* llj.. 15* 16. (continued) B. One-year-old atem inoculated with and killed by Godronia in the field. C. Lesions on 1-year-old atem inoculated with Godronia in the field during April. Typical flagging in fields in which either Phomopala or Godronia were epiphytotic. . . . 66 ......... 66 Wilted branches and discolored leaves A. Typical "flag” or wilted stem. B. Discolored leaves. C. Wilted leaves on a single branch. D. Marginal browning of leaves of affected plant. E. Premature reddening of leaves on affected plants. Apothecia of G. cassandrae f . v acc i n i i ...........70 A. Apothecia on dead 2- to 3-year-old stem collected in late July. B. Lateral view of apothecia (25 X). C. Open apothecia after being placed in a moisture chamber for 2l\. hours. 17. Distribution of ages of wilted stems from which Phomopsis. G odr o n i a . or both fungi were i s o l a t e d ................................... 75 18. Distribution of symptoms in fields in which either Godronia (Godronia Fields) or Phomopsis (Phomopsis Fi eld s) was the only pathogen isolated.......................... 77 19* Distribution of Phomopsis and Godronia in Michigan and Indiana blueberry fields . . . . 20. 79 Final average size of lesions caused by Godronia on inoculated 1-year-old Jersey variety plants after exposure to various temperatures..................................... 91 xiv Figure 21. 22. Page Final average size of lesions caused by Godronia on inoculated 1-year-old plants after exposure to various temperatures.................................. 93 Isolates of (J. cassandrae grown at various temperatures........................... 108 A. Single ascospore isolates from V. corymbosum (a, b ) # single conidTum isolates from corymbosum (c, d), Michigan single ascospore isolate from V_. angustifolium (e), Nova Scotia isolate^ i^rom V. corymbosum obtained from C. L. Uockhart (f), Quebec isolate from V. angustifolium obtained from E. Smerlis Tg) grown at 25 C. B. Same isolates as in (A) grown at 20 C. C. Quebec isolate of f. betlcola obtained from E. Smerlis (aT, Quebec isolate of £. cassandrae obtained from E. S m e r ­ lis (b), Michigan isolate of f. spiraeicola (c), Michigan isoTate of cassandrae (d) grown at 25 C. D. Same isolates as in (C) grown at 20 C. E. Response of single ascospore isolate from V. corymbosum to temperature. 23• Variation in cultural morphology of single ascospore isolates of G. cassandrae f . vaccinii from V. c o r y m b o s u m .................. 110 21].. Asci and ascospores of G. cassandrae f . vaccinii........... A. Asci mounted in lactophenol and stained with 0.1$ cotton blue (800 X ) . 5. Single ascospore mounted in lacto­ phenol and stained with 0.1$ cotton blue. C. Ascus mounted in water (800 X). D. Section cut through pycnidium (800 X). xv 112 Figure 25* Page Conidia of G. ca aaa n d r a e . . ................ lllj. A. Conidia from pycnidium on Earliblue variety (800 X ) . B. Conidia from pycnidium on Earliblue variety (2000 X ) . C. Conidia from pycnidia on Jersey variety (800 X). D. Conidia of f. apiraeicola (800 X). 26. Number of sections infected with Godronia (i.e., number section yielding G o d r o n i a ) when 5 0 0 sections from each group exposed plants were placed on PDA ............ 119 27. Amounts of precipitation recorded at weather stations near Field A (upper) and Field B (lower) between 1 July and 9 October, 1968 .............................. 28. 29. 120 Transverse sections of healthy and Godronia infected blueberry s t e m s ....................... 131 A. Noninfected 1-year-old stem (2f>0 X). B. Incipient necrosis (b) below stoma (a) in center of Type 1 lesion (200 X ) . C. Hypha in air channel of cortex (800 X ) . D. Hyphae in air channel of cortex near reddened cortex parenchyma (J4.OO X). E. Hypha in air channel near reddened cortex parenchyma (800 X ) . Sections of Godronia infected stems ......... A. Radial section showing hyphae in air channel of cortex (800 X ) . B. Hyphae growing along living cortex parenchyma cells (800 X). C. Transverse section through lesion Type 3 showing hyphae in dead cortex parenchyma (800 X ) . D. Transverse section through lesion Type 3 showing deposits in cortex parenchyma (200 X). xvi 133 Figure 29• 30. 31* Page (continued) E. Section through center lesion Typ0 2 showing general necrosis and hyphae (arrow) in air channel (200 X). F. Longitudinal section through pycnidium of Godronia in necrotic cortex tissues (200 X ) . G. Radial section showing hyphae in ves­ sel of discolored xylem of wilted stem (800 X ) . H. Trasnverse section through discolored xylem of wilted stem showing brown deposits (a) and hypha (b) in vessels (800 X). Sections of diseased blueberry stems............ 135 A. Hyphae in necrotic tissue of callus (800 X ) . B. Hyphae and necrosis in bud tissues (200 X ) . C. Disarranged vascular tissue in 8-D type callus. D. Brown deposits between 2 vessels in discolored xylem of wilted stem (800 X). E. Hypha growing through scaliform p e r ­ foration plates of vessel in dis­ colored xylem of wilted stem (800 X ) . F. Brown deposits in discolored xylem of wilted stem (200 X). G. Possible tyloses in vessel of di s­ colored xylem of wilted stem (800 X). Diagram of the probable disease cycle of Godronia canker and stem b l i g h t .............. llj.6 xvii t INTRODUCTION Several Fungi including Coryneum microatictum Berk, and Br. (71*73) Godronia caaaandrae Peck f . vaccinii Grovea (16, 37)* Diaporthe (Phomopala) vaccinii Shear (66, 67), Botryoaphaeria cortioia (Demaree & Wilcox) Arx & Muller (25, 58), and B. dothldea (Moug. ex Fr.) Cea. & deNot. (68) were known to cauae cankers and/or blights of the highbush blueberry (Vaccinium corymbosum L . ). Overgrowths, tumors, or galls on blueberry were reported caused by a Phomopsis distinct from vaccinii (7)* Agrobacterium tumefaciens Sm. & Town (21), and an unknown causal agent (69)• External symptoms observed on dying blueberry stems in Michigan included those caused by each of the above organisms. In addition, C_. microatictum, G. caaaandrae f. vaccinii. Phomopais sp., Fusarium s p p . and bacteria were isolated from 3tems with each of the symptoms reported to be associated with the several canker, blight and gall diseases. It was therefore impossible to accurately diag­ nose the cause of dying blueberry stems in Michigan. Fusicoccum canker caused by CJ. cassandrae f . vac­ cinii (16, 37) was reported in Michigan in 196J+ (3) and the disease was known to be widespread. Little was known, ho w­ ever, concerning the disease cycle and pathological histology of Fusicoccum canker. 1 2 The major objectivea of this research were to: a) determine the primary causes of canker and stem blight of blueberries in Michigan and to define symptoms associ­ ated with each organism; b) develop sufficient information concerning infection periods and sites of infection by Qodronia to provide a basis for fungicide evaluation experi­ ments; c) outline pathological histology during development of cankers caused by Q. cassandrae f . vaccinii. LITERATURE REVIEW Introduction; Modern varieties of cultivated high­ bush blueberries consist of over $ 0 interspecific hybrids developed since the early 1900's. Most varieties are derived from crosses among 7 original selections from the wild and include genes from V_. australe Aiton, V. larmarkii Camp, and V. corymbosum. For consistency, all varieties will be referred to in this thesis as V. corymbosum even though the genomes of some common varieties grown in Michigan such as Jersey consist entirely of V. australe genes (3 8 ). All blueberry literature was reviewed recently and only pertinent disease literature will be discussed (2 3 ). Stem blight and die-back diseases of Blueberr ies : Stem blight or die-back diseases of the highbush blueberry are caused by P_, vaccinii Botrytls cinerea Deb. (6 6 , 6 7 ), B. dothldea (68), (61), Monillnia vaccinli-corymbosl (Reade) Honey (61), and Glomerella cingulata Spaulding & von Schrenk (61). Botrytis causes a twig and blossom blight of blue­ berries which is serious only in areas where cool, rainy conditions prevail (61). Certain symptoms of Botrytis twig blight can be confused with those caused by cold injury, B. dothidea, and vaccinii (68). 3 The disease is generally confined to succulent shoots, blossoms, occurs wherever blueberries are grown and fruit and (61). Monilinia and Glomerella can infect young stems, but twig blights caused by these fungi are not important (53, 61). Phomopsis twig and stem blight was reported in North Carolina, Scotia (36). New Jersey, Massachusetts (66), and Nova The disease was first reported by Stevens °(6l) and later described by Wilcox (66). Wi lco x (6 7 ) established that the blueberry pathogen was D. vaccinii which also causes a cranberry fruit rot. Wilcox (66) reported that black lesions developed on wounded or nonwounded succulent shoots within 2-3 days after artificial inoculation at 70-76 P. Lesions expanded and the organism eventually grew down inoculated shoots to older stems which were "girdled" within a few weeks. Dead portions of 3tems were sharply demarcated from living sec­ tions. Such symptoms resembled cold injury (61). Localized lesions developed when older stems were inoculated directly. Leaf spots developed on leaves 2 months after spray inocu­ lated plants were placed in a cold frame Wilcox (6 7 ). (66) concluded that Phomopsis infects and grows down succulent shoots to older stems which are "gir­ dled." Varney and Stretch (61) reported that older stems are frequently "girdled" when the crown is infected by the fungus. Leaves on "girdled" stems wilt and turn brown d u r ­ ing hot weather (66). The meaning of "girdle” was vague in the reports of Wilcox (66) and Varney and Stretch (61), They reported that blight resulted when Phomopsis "girdled" stems, but it was not clear whether this meant that stems were cankered, vascular tissues were blocked, or both. Other investiga­ tors (3 6 , 73) however, have mentioned that cankers were associated with vacclnil. According to the literature, Phomopsis twig blight is a disease of weakened bushes and is a minor disease of blueberries (20, 61, 66). Sound cultural practices and eradication by pruning are the only recommended control measures (61). It is not known when infection occurs although symptoms on young shoots were normally observed in the spring. Pycnidia were found on leaves and overwintering twigs, but the time of year when observations were made was not mentioned (66). Pycnidia were observed in February on a plant inoculated in July. It was not indicated whether plants were grown in the greenhouse or outside or if plants were observed between July and February (66). Pycnidia occurred on dead stems during August in Nova Scotia (3 6 ). Perithecla of D. vaccinii form on cranberry fruit (6 7 ), but are rare on any blueberry tissues. Lockhart (3 6 ) observed perithecia on stems of V. angustifolium and V. corymbosum in Nova Scotia. This is the only report of Dlaporthe perithecla on blueberries. 6 Blueberry 3tem blight caused by B. dothidea was serious in North Carolina. Yellowing, wilting, and brown­ ing of leaves on affected branches were the most conspicuous symptoms of the disease. Xylem of affected stems turned pecan brown and branches with browned leaves were often in close proximity to branches with healthy leaves (68). Similar symptoms have been observed on blueberries in Michigan. Blueberry stem blight is probably confined to the south (61) although a single report of a Botryo3phaeria occurring on blighted stems of blueberries collected in Ohio, Michigan, New Jersey, and Illinois was published (1*4) . Canker diseases of blueberries; Several canker diseases of the highbush blueberry have been serious in North Carolina (9, 25, 55)» Nova Scotia (16, 35)* Massa­ chusetts (70, 73), Washington (31)» British Columbia (37), and Michigan (3)* Blueberry stem or cane canker caused by B_. corticls prohibits cultivation of certain blueberry varieties in North Carolina (9, 25, 55)* Except for a single report of the disease in New Jersey (61), stem canker does not occur in the north. Fusicocoum canker caused by G. cassandrae f . vaccinii (16) occurs in Quebec (11), Nova Scotia (13, 16), British Columbia (12, 37), New Brunswick (16), Washington (25, 31), Michigan (3), Maine (16), Finland (30), Holland, and England (3). The disease is considered to be the 7 factor limiting increased cultivation of the highbush blue­ berry in Nova Scotia (35?) and Washington (31)» The disease was first reported in 1931 - (1 1 ), but few definitive studies have been published. The asexual state of G. cassandrae f . vaccinii was isolated from rotting cranberries and described as P. putrefaciens by Shear (I4.6 ). Shear and Bain (I4.7 ) later showed P. putrefaciens to be the imperfect form of G. cassandrae. a Discomycete described on leatherleaf (Chamaedaphnae calyculata (L.) Moench) by Peck in I887 (14-2 ). The imperfect state of G. cassandrae was first iso­ lated from cankered blueberries in 1931 (11).. In 1958, Creelman (16) and McKeen (37), reporting independently, called the disease Fusicoccum canker and blueberry canker, respectively. Although both found apothecia of Godronia on diseased stems, J. W. Groves, in a communication to Creelman (16), indicated that the organism on Vaocinium was not the same as the one on Chamaedaphnae and that the former was probably undescribed. However, following a thorough study of the genus, Groves (28) indicated that the organ­ isms inhabiting Chamaedaphnae and Vaooinlum were the same 3pecies and that Godronias on Be tul a. Spi rae a, C alluna, and Ribes were also morphologically indistinguishable from G. cassandrae. Groves therefore tentatively erected several forms of G. cassandrae based on the genera of plants on which the fungi were found (Table 1). 8 Table 1. Godronia cassandrae as interpreted by J. W. Groves (28). Forms of Godronia cassandrae Host genera G. cassandrae f . cassandrae Chamaedaphnae it ti f . beticola Betula it it f . callunae Calluna it it Ribes it it ti it . ribicola f . spiraeicola f . vaccinii f Spiraea Vaccinium It must be stressed that this was not a formal taxonomic treatment and according to Groves (28): The taxonomic status of the forms described,..is some­ what doubtful.,..- In view of the pathogenic signifi­ cance of the Vaccinium fungus, it was felt desirable to maintain some sort of distinction between the strains occurring on different hosts and for consistent treat­ ment it was decided to designate them as forms. Groves (28) indicated that Fusicoccum is an u nac ­ ceptable name for asexual states of Godronia because they are morphologically dissimilar to the description of the type of the form genus Fusicoccum. Groves felt that Topo- spora was the earliest acceptable name for macroconidial states of G o d r o n i a . In 1968, Smerlis (5>1) reported that isolates of G. cassandrae from several ericaceous shrubs (Andromeda g l a u - oophylla Link, £. calyculata, Kalmia angustifolia L., Ledum 9 groenlandicum Oeder, and V^. angustifollum) were pathogenic on all ericaceous shrubs tested regardless of the source of the isolate. In addition, isolates of G. cassandrae from Alnus rugosa (Du Roi) Spreng. var. american (Regal) Pern., Betula alba L., B. papyifera Marsh, B. populifolia Marsh., and Sallx sp. were pathogenic on all salicaceous and corylaceous hosts tested. However, isolates from the second group were not pathogenic on ericaceous shrubs and vice versa. Smerlis therefore placed G. cassandrae found on Ericaceae (other than Vaccinium species) into form cassan­ drae and isolates from the other hosts into form beticola. He also noted that isolates from V,. angustifollum were cul­ turally distinct from isolates from the other ericaceous shrubs, the latter being green with abundant mycelium, whereas those from Vaccinium were black, often slimy, with sparce, grey, aerial mycelium. Smerlis, therefore, placed G. cassandrae on Vaccinium species as form vaccinii Smerlis' (51). (51) interpretation needs further study in light of McKeen's (37) failure to infect blueberries with isolates of Cx. cassandrae f . vaccinii from cranberry. Cankers on 1-and 2-year-old blueberry stems first appeared as small reddish discolorations in the epidermis (16). Such lesions were observed in the winter in British Columbia (37) and late winter to early spring in Massachu­ setts (73)• Incipient lesions expanded rapidly in the spring (3 7 » 7 3 ) and became dark red (7 3 )» black (3 7 )> or 10 brown (16) In color. Such lesions in Nova Scotia had gray centers which turned brown and died (16). coalesced in British Columbia (lj.8 ). Some lesions A "bullseye" pattern of concentric zones of alternating light and dark tissue on 1-and 2 -year-old stems was considered diagnostic in Massachusetts (73) and was reported in Michigan (3)* but not elsewhere (1 6 , 37 )* In Nova Scotia, cankers were up to 3-5 inches long and cankers on actively growing stems caused them to become flattened or depressed. As cankered tissue died, bark sloughed and cankers usually "girdled” infected stems in 1 year (16). Other workers (3* 37, 7^4-) did not discuss canker development on stems older than 2 years. The most striking symptom of the disease was the "flag" or wilted stem. Infected stems began to wilt and die in May in British Columbia (37) and Massachusetts but not until June in Nova Scotia (16). (73)* Wilting, which took place within a few hours on warm, dry days (1 6 ), co n­ tinued throughout the growing season (1 6 , 3 7 , 7 3 )• instance, In one it was reported that the interval between appear­ ance of incipient lesions and cane death was about 18 weeks in Massachusetts (73)- Most infections were centered by leaf scars in British Columbia (37) and occurred at the ground level and even below the soil surface (37) (1 6 ), but some infections also occurred on higher parts of the stem (16, 37)* Creelman 11 (1 6 ) reported that infection progressed from cankered crowns into bases of new stems. Xylem beneath lesions turned brown (16, 73)• C ree l­ man (“1 6 )“reported, "The cortex, cambium, and to a limited extent, the xylem are invaded by the fungus.1' He added, “Discolored areas in the xylem do not extend deeply before death of the stem occurs." No mention was made in the literature of Godronia killing stems without causing can­ kers (3 , 1 6 , 3 5 , 3 7 , 6 1 ). The relative roles of conidia and ascospores in the disease cycle of Fusicoccum canker and when infection occurs are not known (3, 16, 35, 37, 61). Pycnidia were present beginning in late March in British Columbia (37), from midMarch to mid-July in Massachusetts September in Nova Scotia (16). (73), and from July to In Michigan, conidia were trapped with a Hirst spore trap during July, August, and September (3)» Apothecia were abundant in Nova Scotia with suc­ cessive annual crops of apothecia occurring on 3 -year-old pruning stubs and diseased prunings in British Columbia chusetts (73). (1 6 ), but were scarce (37), and were not reported in Massa­ McKeen (37) concluded that ascospore inocu­ lum was unimportant in British Columbia because apothecia were rare. Dispersal of ascospores corresponded with the appearance of pin point lesions in Nova Scotia. It was not stated when this occurred, however apothecia were found in May (16). 12 Although different workers have proposed spring (1 6 ) and fall (3 5 »3 7 ) infection periods, sound evidence in support of either is lacking. Creelman (16) and Fi tz­ patrick and MacSwain (II4-) apparently felt that infection occurred in the spring since they applied protective fu ngi ­ cides at that time. McKeen (3 7 ) reported that "some, if not all" infec­ tion occurred after June in British Columbia since parts of stems which were formed during July and August were diseased. However, he did not state when or how often infections on new stems were observed. Artificial inocu­ lations made in November and December caused cankers whereas those performed in June and July did not. Conidia were reported to be most abundant in the spring and ascospores were considered to be unimportant. McKeen concluded that infection by Godronia probably occurred during late summer and fall rains. Lockhart (35) concluded that infection occurred in the fall because inoculations were successful during Octo­ ber, May, and August, but not during July "when plants were growing vigorously." He added, "The successful inoculations of August 28 and October 2 coincided with slowing down or cessation of growth." He implied that inoculations made in May which did not cause visible cankers until late August supported this conclusion. 13 In Massachusetts, Zuckerman (73) obtained infection on plants inoculated in a screenhouse during March and April, but did not speculate on when infection occurred in the field. It is clear that existing data did not tell us when Godronia infects blueberries. Several workers have reported apparent differences in field susceptibility of blueberry varieties to Fusicoccum canker. Data published on susceptibility of varieties of blueberry are summarized in Table 2. No effective control measures have been developed, although Lockhart (38) and Nelson (39, 4°) have reported limited control when compounds such as phenyl mercury acetate were applied in the fall. Cultural characteristics of CJ. cassandrae varied considerably depending upon source of isolates and culture media. Shear (lj.6 ) reported that isolates from cranberry varied in color from shades of yellow-green and yellow to pink and brown depending upon the culture medium. McKeen (3 7 ) indicated that isolates from cranberry and blueberry differed in color and colony texture on a range of media. He also stated that single conidial and single ascospore cultures of the isolates from blueberry differed from one another. He noted that G. cassandrae f • vaccinii varied from brown to buff-pink to grey. Cranberry isolates had more aerial mycelium and sclerotia were produced by single IllTable 2. Susceptibility of varieties of blueberry to infection by Qodronia cassandrae f . vaccinii. Variety Susceptibility® rating Reference Atlantic - 14 Berkley H 35 Blueray H 35 Burlington H 15, 35 Bluecrop H 35 Coville H 35 Cabot - 73 Concord R 35 Jersey Hb 3, 14, 15, 35, 48, 73 Earliblue H 35 Johnson H 35 Pemberton - 3, 14, 73 Pioneer H 73 Rub el R-L 3, 37, 73 Rancocas R-L 15, 35, 37 Stanley M 3, 35 H = high susceptibility; M = moderate susceptibility; L - low susceptibility; R = resistant; - = infected, but no data given on relative susceptibility. b Jersey was usually considered to be most susceptible and Rancocas most resistant. \ 1$ aacospore and condial isolates from blueberry (37)• lis Smer­ (5 1 )* as mentioned earlier, also noted differences in colony texture and color when cultures of forms cassandrae and vaccinii were compared. Gremmen (27) reported that colonies were gray at first becoming gray-green to yellowgreen. Groves (28) stated that cultural characters are quite variable and that even spore morphology can vary ’’depending upon the preparation.” Groves (28) also reported that microconidia were common both on the host and in cul­ tures of G. cassandrae. Godronia cassandrae grew at temperatures as low as O-I4. C (16, 37) • McKeen (37) reported that maximum growth occurred at 20 C on potato dextrose agar and growth was inhibited at 30 C. At J4. and 10 C, growth was $0 and 60$ of the maximum, respectively (37)• Stevens (52) reported that cranberry end rot caused b y G. cassandrae f . vaccinii developed well at 0 C and 15-20 G, but the lower tempera­ ture was more favorable for development of rot. Coryneum canker caused by Coryneum microstictum Berk. & Br. was reported in Massachusetts by Zuckerman (7 1 ) who later demonstrated pathogenicity of the organism on blueberries (73)• Acervuli of the organism were common on sun scald areas and well developed cankers were sunken in appearance with acervuli on the surface. Cankers expan­ ded until stems were completely "girdled” and parts of the stem above the "girdle" died. In addition to jC. miorostictum, 16 P. vaccinii was also isolated from several branches with symptoms of Coryneum canker (73)* Based on field and inoculation studies, Zuckerman (73) concluded that Coryneum affected only weakened plants. Coryneum canker has not been reported elsewhere (61). Other organisms which are known to cause cankers of blueberries either are not important or are confined to areas of limited cultivation. Bacterial canker caused by Pseudomonas syringae Van Hall was reported in British Colum­ bia (37)i Washington and Oregon (62), but not elsewhere. Overgrowths, tumor, and gall diseases of blueberries; Overgrowths of various types on stems of blueberries were reported caused by A. tumefaciens (21), Nocardla vaccinii Demaree and Smith (22), Pucciniastrum myrtilll Arth. (61), and Phomopsis s p . (7)* (Schum.) A root gall disease was described (6 9 ), but the cause is not known. As the name indicates, bud proliferation gall caused by N. vaccinii, is characterized by extensive bud proliferation in the crowns of infected plants (22). The disease is not economically important and is distinct from symptoms observed in Michigan. A witches broom caused by P^. myrtilli has been found in Michigan (39), but the disease is distinctive and extremely rare in the state. In 1938, Brown (7) reported that a Phomopsis, di s­ tinct from £. vac cinii, caused galls on inoculated 17 blueberries. She reported the disease in Massachusetts, New Jersey, Oregon, and Michigan. The gall symptoms pro­ duced on inoculated blueberries were not striking and as Varney and Stretch (61) indicated, "...were more indicative of cankers than galls." Calluses developed on Jasmium nuldflorum and Viburnum opulum more quickly and more exten­ sively than on blueberries (7). Brown also reported Phomopsis galls on Ulmus amerlcana (8 ), Acer s p . (8 ), Privet (8), coral berry (8), Forsythea s p . (8), Quercus s p . (6), Viburnum (fj), and Fagus s p . (6). Her results on blueberry have never been repeated (5^-)* Demaree and Smith (21) reported that tumors iden­ tical to the Phomopsis galls described by Brown (7) were induced by isolates of A_. tumefaciens obtained from blu e­ berries. The disease was observed in New Jersey, New York, Michigan, Washington, and British Columbia. Most galls were on branches and small twigs, but some were at bases of canes near the ground line. roots. Galls were not found on Affected buds died and galls were perrenial. Detailed studies of galls indicated that the causal organ­ ism was a strain of A. tumefaciens distinct in host range and in cultural characteristics from A. tumefaciens on apple and peach. Only A_. tumef aciens isolated from blue­ berry was pathogenic on that host and the bacteria p r o ­ duced large galls on inoculated plants. Apple and peach strains, although infective on their respective hosts, did 18 not affect blueberry, confirming Brown's similar isolates. (7) results with Phomopsis was not associated with the tumors and none of the fungi found in galls caused callus formation on inoculated blueberries (21). A gall disease described in 1956 by Zuckerman (69) as a new root-gall disease was caused by an unknown agent. The disease was characterized by galls on all woody portions of the plant. Zuckerman described galls as being either white and coriaceous or dark brown, woody and covered with bark. Long basal cankers were often associated with galls. According to Zuckerman (69), death of affected branches was not due to the "girdling" action of the cankers. supporting this conclusion were presented. No data He further reported that some plants had galls on the roots, but not on aerial portions. Histological sections revealed no hyphae or nematodes in gall tissue. When ground gall mater­ ial was injected into Pioneer variety stems, leaves wilted within 3-5 weeks after inoculation and stems died within 3 months. lation. Adjacent stems died during the year after inocu­ Gall and canker symptoms did not appear on inocula­ ted plants. No pathogenic organism was isolated from affected tissue. Pioneer, Cabot, and Wareham appeared to be very susceptible, but Jersey, Rubel, and Dixi appeared resistant. Zuckerman (69) concluded that the disease was not crown gall since symptoms differed from those of A. tumefaciens affected plants. He cited, for example, that 19 galla did not occur on roots in Demaree and Smith's (21) study. The size and position of tumors on affected stems differed from crown gall tumors and cankers were not associated with crown gall tumors studied by Demaree and Smith (21). In Michigan! the symptoms associated with dying stems of blueberry plants overlapped those reported for Fusicoccum canker (16, 37, 73)» Coryneum canker (73), Phomopsis twig blight (61, 66, 6 7 ), Botryosphaeria stem blight (68), Phomopsis gall (7), crown gall (21), and root gall (6 9 ). Phomopsis vaccinii, G. cassandrae f . vaccinii, and J3. microstictum were associated with the symptoms in the field (614.) • PART I. SYMPTOMATOLOGY. Methods and Materials. Association of organisms with symptoms; Blue­ berry stems with canker or blight symptoms were collected from various locations in Michigan and Indiana over a 3year-period. All stems were coded according to symptom, variety, date, and location of sampled fields. Preliminary investigations indicated that symptoms could be categorized according to the scheme of symptoms illustrated in Figures 16. All symptom data are based on these categories. Early observations showed that growth of Godronia from diseased tissues was significantly reduced if stems dried or were exposed to temperatures exceeding 25 C for more than hours. Stems were therefore stored at 10-12 C in tightly wrapped plastic bags after they were collected from the field. Stems were cut into 1-2 cm long sections, surface sterilized in 1.3# sodium hypochlorite plus 2-3 drops Tween 20 surfactant for 1 minute, and placed on (see Table 3 for descriptions of all media used in this research) 15-20 ml h PDA (pH = 5*6-6.8) in plastic petri plates. Half strength PDA was used because preliminary investigations showed that G. cassandrae f . vaccinii sporulated more profusely on 20 21 DPA than on PDA or ^ PDA. All cultures were grown in the dark at 10-12 C for 6-18 weeks. had several advantages. mum temperatures. Growing cultures at 10-12 C Godronia grows slowly even at opti­ Maintaining isolation plates at 10-12 C allowed ample growth of God ro n i a , Phomopsis, and Coryneum while minimizing overgrowth of cultures by common sapro­ phytes. Sections could be plated and held in the refrigera­ tor for several months. Large numbers of stems were col­ lected, sectioned, and plated during the summer and observed as time permitted. and identified. Commonly observed fungi were isolated The same procedures were used when reisola- ting fungi from inoculated tissue. Table 3 . Ingredients and abbreviations of media used in this research. Medium Potato dextrose agar Abbreviation PDA potato dextrose agar PDA Ingredients/ liter 39 g Difco potato dextrose agar 20 g Difco PDA, 8 g agar Malt agar MA g Difco malt agar Nutrient agar NA 23 g Difco nutrient agar Dextrose, nutrient, peptone, yeast agar DNPYA 20 g PDA, 20 g NA, 5 g Difco bacto pep tone, 8 g agar, $ g Difco bacto yeast extract. 22 Isolation of organiama from diseased t iss ue: Mono- conidial isolates of fungi were obtained in one of the f o l ­ lowing ways: a) spores were harvested directly from a sporophores with a sterile needle; b) single spores were picked from the surface of agar after streaking suspensions of conidia on the agar surface; c) single colonies of fungi were harvested after spores from a suitable dilution series had germinated and grown in nutrient media. Single ascospores of Godronia were harvested from from the surface of a thin layer of agar in plastic petri plates after spores had been ejected from inverted apothecia fastened to the lids with masking tape. Hyphal tip isolations were necessary when fungi did not sporulate. All isolates were transferred initially to PDA acidified to pH 3*5 with 10# lactic acid in order to m i n i ­ mize bacterial contamination. all fungi were grown on Following initial Isolation, PDA (pH = 5•6-6.8) at 22-25 C in the laboratory and were transferred every Zj.-6 weeks. Bacteria which grew from diseased tissue were sus­ pended in a suitable volume of sterile distilled water and streaked on NA or DNPYA (pH = 6.8). Single colonies were then transferred and grown on the same media at 22-25 C. All bacteria were transferred weekly. Inoculations: Depending upon the experiment, 1“ or 3-year-old Jersey, Blueray, or Bluecrop variety blueberry 23 plants were inoculated. All 3-year-old plants were grown out-of-doora in field soil contained in 12 quart galvanized pails. One-year-old plants were grown in 1:1 (v/v) mixture of Michigan peat and sandy loam contained in 8-inch clay pots. Soil pH was not determined. One-year-old plants were transplanted from cutting beds as 7-month-old cuttings and grown at 23-30 C under continuous light (daylight supple­ mented with two 500 watt incandescent bulbs/bench) in the greenhouse until large enough to inoculate (Pig. 8 - A ) . All plants were fertilized bimonthly with 2 $ ml of a solu­ tion containing 1 tablespoon Plant Marvel (12, 31> and 11+$ N, P, K, respectfully) / gallon tap water. Inocula consisted of fungi from l+-week-old cultures or bacteria which were transferred daily for 1 week prior to inoculations. After swabbing stems with 9$$ ethyl alco­ hol, water suspensions of conidia, mycelium in blocks of ^ PDA, or a single inoculating loop of bacteria were placed on the stems. Depending upon the experiment, the inoculum and the stem tissue were pierced 1-2$ times with a sterile needle. In some studies, the inoculum was inserted under 1.5 cm long V-shaped flaps cut into the bark or epidermis. Following wounding, the inoculation sites were wrapped with sterile, moist cotton and wrapped with plastic film held in place with rubber grafting strips (Pig. 7-A ). The number of inoculations per plant varied from 1-10 depending upon the experiment. All plants were placed in a mist chamber (18-25 C, 90% + relative humidity) for 5-10 days. Cotton and plas­ tic were removed and the plants were transferred to a green­ house and grown at 15-20 C under continuous light after the 5-10 day incubation in the mist chamber. Controls con ­ sisted of: a) separate plants inoculated 5-10 times with blocks of PDA or sterile distilled water and b) single control inoculations on each inoculated plant. 25 Pig. 1. Lesion-young canker infection type on 1- and 2-yearold stems. All lesions were caused by G o d r o n i a . A) Incipient lesions (red spots) on 5-month-old stem collected fr om an upper Michigan field 3 December, 1968. B) Lesions of various sizes on a 1-year-old stem collected from an upper Michigan field 15 June, 1967. C) Lesion at the base of a ij.- to 5-month-old stem collected from a northern Michigan field 15 O c t o ­ ber, 1965* Note the leaf scar at the center of the lesion and the absence of pycnidia. D) Typical lesions on 1-year-old stems collected in mid-summer. Note the split epidermis and the lesion at the base of the lateral on (a) and the nbullseye" pattern of the lesions with pycnidia on (b) and (c). 27 Pig. 2. Lesion-young canker Infection type on 1- and 2year-old stems. All stems infected with Qodronia. A) Lesion on 1-year-old stem collected from an upper Michigan field 15 June, 1967* Note the pycnidia. Split in the center is atypical. B) Typical "bullseye” lesion with pycnidia on a stem collected in late May. The red-brown to maroon coloration is common. C) Young canker on a 1-year-old stem collected in July. Note the depressed center and dead epidermis and that the canker is wide in rela­ tion to its length. A few pycnidia are visible. D) Young canker on 2-year-old stem collected 15 October, 1965* Note the leaf scar at the cen­ ter and that the canker is wide in relation to its length. Smooth, tan colored epidermis (a) was considered to be caused by sun scald. 28 29 Pig. 3 . Developed canker infection type on older sterna. All cankers were caused by Go dro n i a . A) Cankers and depression on 3 to i^-year-old stems. The gnarled appearance (b) and can­ kers which were wide in relation to their length (a) were common on older stems infec­ ted with Godronia. B) Developed canker on l4.-year-old stem. Note that the bark is dead and flaking, the hint of cal­ lus at the edges of the canker and the leaf scar near the center. C) Mild expression of callus type 8-E. Small internodal lesions (Pig. 1-B) observed in April were often apparently walled-off by August ap shown here. D) Callus type 8-E. This symptom was similar to calluses formed when plants were mechanically wounded. Note the smooth, rounded edges of the callus and the pycnidia at the center. 31 Pig. ij.. Callused blueberry stems. A) Callus type 8-A on 3-year-old stem of a Stanley variety plant. This type of callus was at the bases of lateral branches and there were always cankers at the base of the callus. B) Callus type 8-A on Pemberton variety. C) Callus type 8-B on Earliblue variety. The cause of this type of callus was not proven. Note the corky appearance and that the callus extends the length of the affected stem. Cankers (a) were usually found associated with 8-B calluses. D) Callus type 8-B on Earliblue variety. E) Photograph off crown gall on blueberry provided by the Canadian Department of Agriculture. Note the similarity to the 8-B calluses in C and D. 32 33 Pig. Callused blueberry stems. Godronia was isolated from stems in A, B, D, and W. A) Callus type 8-C on Jersey variety. This type of callus was characterized by being located in crowns of affected plants. The callus was often concentrated along edges of large cankers. B) Callus type 8-C. Note that calluses occur at the bases of each lateral originating from the cankered crown. C) Crown gall (?) symptoms on blueberry in Michigan. Photograph supplied by J. W. Nelson, Michigan Blueberry Growers Association. As far as could be determined, diagnosis was based on symptoms only. D) Callus type 8-D. Note that the callus occurs along edges of a canker located above the crown. E) Callus type 8-D. Note the large size of the cal­ lus and that it occurs along the upper edges of a canker (a) located above the crown. 34 * 35 Pig. 6 . v Miscellaneous symptoms associated with blight and canker diseases of blueberry in Michigan. Godronia was isolated from each of the stems. A) Discolored bark or epidermis infection type on unnamed variety photographed in May. B) Split or flaking bark or epidermis infection type on 3-year-old stem photographed in late June. Note the erumpent pycnidia of Godronia concentrated in the bark fissures. C) Split or flaking bark or epidermis infection type on 2 to 3-year-old stems. Note the depres­ sion (a) and the red-brown discoloration (b). D) Discolored bark or epidermis infection type on 1-year-old stem of an unnamed variety ph o t o ­ graphed May 29, 1968. Note the sporulating pycnidia. E) Discolored bark or epidermis infection type on a 2-year-old stem photographed in May. 36 Experimental Results Association of organisms with symptoms: The genera of fungi identified on diseased stems are shown in Tables lj. and 5« Only those fungi which occurred on more than 2.0# of sections observed are discussed. Alternaria s p p ., Epicoccum s p ., 13. cinerea, and Papulospora sp. were considered to be saprophytes and unim­ portant in canker or blight etiology. Alternaria and Epicoccum are common saprophytes on Vaocinium s p p . in Massachusetts (65, 72), New Jersey (52, 65), North Caro­ lina (65), Washington (25), and Wisconsin (26). Botrytis causes a twig and blossum blight of blueberries (61), but the association of Botrytis with diseased stems was infre­ quent until the cool, rainy 1968 season. Papulospora s p p . are generally secondary invaders of diseased tissue and have not been reported on blueberries (56)• Papulospora was generally associated with weakened tissue and espe­ cially with old, weathered calluses. Although Fusarium s p p . have not been reported on blueberries before, species such as I?, solani and F. oxy- sporium are known to cause cankers (I|., 3k-» and overgrowths (1) on other woody plants. 56, 57) Fusarium s p p . isolated from cankered and callused blueberry stems did not cause disease when used to artificially inoculate healthy plants. 37 38 Table Ij.. Fungi isolated from stem sections taken from cankered and/or blighted blueberry plants.® Fungi*5 % sections with fungus Godronia cassandrae f. vaccinii 14.0 . 0 Diaporthe (Phomopsis) vaccinii 27.0 Coryneum microstictum 2.9 Fusarium spp. 2.0 Botrytis cinerea 6.0 Alternaria spp. llfl.il. Epicoccum sp. 27.2 I4..6 Papulospora sp. Coniothyrium sp. < 2.0 Verticillium sp. < 0.1 Phoma sp. < 2.0 Dendrophoma sp. < 2.0 Melanospora sp. < 0.1 Pullularia spp. < 2.0 Sphaeronema sp. < 0.1 +Bispora sp < 2.0 +Cephaiosporium sp. < 0.1 +Cylindrocarpon sp. < 0.1 Pyrenochaeta sp. < 0.1 unidentified <3.0 a Based on 3130 sections collected during 1966-68. k+identification tentative. 39 Table £. Sporu.latj.ng fungi identified on sections of diseased blueberry stems placed in moisture chambers.a Fruiting type Sexual Asexual Fungi Glomerella cingulata + Nectria cinnarbarina - + Festalotia sp. ** + Penicillium spp. *• + Trichoderma spp. *» + Sordarid spp.*. - + Tympanis sp. Chaetomium sp. 9 • - a Godronia and Pliomopsi s also sporulated in moisture chainbers, but these data are not presented. k + = observed; - = not observed; ? = not determined. 14-0 The asaociation of Godronia. Phomopais. and Coryneum with aymptoma obaerved on diseaaed blueberry atema ia shown in Table 6 . Each organism was associated with all of the symptom categories, but Coryneum was relatively uncommon* Table 6 . Association of canker fungi with symptoms observed on blueberry stems. Percent sections with funei Godronia Phomopsis Coryneum Symptoms Total sections None 20.0 38*i+ 2.0 2I4.2 Lesion-young canker 65.0 8.1 0.8 '14.06 Developed canker 32.9 27-9 1.9 670 Callused canker 71.2 13*3 3*2 2k7 Discolored bark or epidermis 22.5 2 2 .ij. lj..7 695 Split or flakbark or epidermis 39.6 36.6 2.6 870 Isolates of Phomopsis from diseased .blueberry stems collected in Michigan and Indiana had the following char­ acteristics. Single oCconidium isolates grown on were shite to grey in color. FDA Colonies were velvety in tex­ ture with concentric zones of abundant and sparce aerial mycelium. All isolates attained growth at 25-30 C (Fig* 9). 1*1 Isolates obtained from blighted stems collected in New Jer­ sey were similar except that they were black in color. Only Q(conidia were produced by single C k conidium isolates when they were grown on PDA or PDA. Alpha conidia were hyaline, oyoid, biguttulate, and measured 6-8 x 3 (2). Ju in lactophenol Bete conidia measured 1.0 x l£-20 Pycnidia on naturally infected stems varied as to the type of conidia produced. Some pycnidia produced only o< conidia or only ^ conidia whereas others produced both types. Production of dL and conidia was affected by light t culture media (6 7 )* temperature (3 3 » 6 0 ), and host sub­ strate (I4.I) in studies of other species of Phomopsis. In this research single o i conidium isolates from pcynidia p r o ­ ducing bo th spore types produced only oC conidia in culture. Production of both ei and £ conidia was not considered a suitable jharacter for positive identification of Phomopsis isolated from blueberry. Therefore isolates were consid­ ered to be Phomopsis when they produced c k conidia and col­ onies similar to those produced by single conidium iso­ lates fro m pycnidia producing both spore types. That this Phomopsis was P. vaccinii was determined by pathogenicity on bluebqrries, cultural characteristics and spore morphology. Characteristics of G. cassandrae f. vaccinii iso­ lated frc m blueberries will be discussed in Part II of this thesis. U2 Cultures of Coryneum were not studied in detail, however, single conidium isolates produced gray to brown aerial mycelium on hz PDA. 12 week old cultures. Acervuli were produced on 10- Conidia were 3-1+ celled, honey colored, and measured 17-20 x (Pig. 8-C). 8-8.1 fx in lactophenol These observations are in accord with Zucker- man's (73) description of _C. microstictum isolated from blueberries in Massachusetts. Inoculation of plants in the greenhouse; Only Godronia and Phomopsis were pathogenic on 1-year-old Jer­ sey, Blueray, and Bluecrop plants inoculated with Godronia, Phomopsis, and Coryneum (Table 7)* There were no differ­ ences in response of the 3 varieties to a particular pathogen. Symptom development was followed on the inocu­ lated plants for 10-2ij. months. Table 7» Results of artificial inoculations of blueberry plants with isolates of Godronia, Phomopsis, and Coryneum. Number isolates tested Fungus Number of inoculations % inoculations causing disease Godronia 8 183 82.3 Phomopsis 5 125 52.0 Coryneum 2 8 0 Both Godronia and Phomopsis caused lesions on inoculated 6 month to 1-year-old branches. Lesions caused k3 by Qodronia were dark colored at first (Fig. 7-C) and turned red-brown to brown (Fig. 7-D# 8-B, 11-D). Bands of water-soaked and reddened tissues were often apparent at the periphery of necrotic tissues (Fig. 7-D). The centers of lesions turned gray after a few weeks due to death of cortex and epidermis (Fig. 8-B). Pycnidia devel­ oped on lesions within 2-3 weeks and continued to appear for an additional I4.-7 weeks when plants were grown under continuous lights in a 15-20 C greenhouse (Fig. 7-D). Inoculated branches wilted within 7 weeks (Fig. 7-1)# but fewer than 1% of branches inoculated with Godrotfla wilted under these conditions. within 1+.-7 weeks. The lesions attained maximum size Outer tissues died and sloughed off 10- 15 weeks after inoculation (Fig. 7-E). As greenhouse temperatures increased during the spring, cankers appeared to become localized by the host. Such cankers did not expand when plants were transferred to a cold frame and grown outside for 1 year (Fig. 10-C). Lesions caused by Phomopsis developed more rapidly than those caused by Godronia and by the 10th day after inoculation (Fig. 7-H) succulent shoots were wilted. Lesions on 6 month to 1-year-old stems were darker and larger than Godronia lesions of a comparable age (Fig. 7-F, G). Lesions on succulent shoots coalesced and large areas on the inoculated stems were necrotic within 10-15 days after inoculations (Fig. 7-H). Pycnidia developed in ¥4- Pig. 7- Inoculation results. A) W rap ped inoculation site. B) Control inoculation. C) Response on Jersey variety 10 days after inocu­ lating w ith G. caasandrae f . v a c c i n i i . The pl ant was grown Tn the 15 C mist chamber f oll owing inoculation. D) Response on Jersey variety 9 weeks after inocu­ lating w ith G. caasandrae f . v a c c i n i i . Note the pycnidia and the discoloration in advance of necrosis. The plant was gr own in a 18 C greenhouse. E) Canker on Jersey v ariety 11 months after in ocu ­ lating w it h Cr. caasandrae f . v a c c i n i i . Note the similarity to Pig. 2-C. The plant was grown in a cold frame f oll owi ng inoculation. F) Response on B lue c r o p variety p l a n t 10 days after inoculating with £. vaccinii (b). Control inoculation is at (aTT The pl ant was grown in the 18 C mist chamber. G) Lesion on Bluecrop variety plant 10 days after inoculating with £. v a c c i n i i . H) Young shoots which w ilted 10 days after inocu­ lating w i t h P\ v a c c i n i i . Plant was grown in the 18 C mist c h a m b e r . I) Jersey variety stem wilted 6 weeks after inocu­ lating w i t h G. caasandrae f ,v a c c i n i i . The arrow indicates the inoculation site. The plant was grown in a 18 C greenhouse following inocu­ lation . k5 U-& Pig. 8. A) Typical plant inoculated in studies per formed in the greenhouse. B) St em naturally infected w i t h Go dr o n i a (a) and a r t i ­ f i cia ll y inoculated stem (b). Note the gray-brown centers and maroon colored edges of the lesions. C) Section through acervulus of C. microsticturn on a b l ue berry stem (ij.00 X ) . Section was cut w ith an experimental microtome (2 9 ) and m o u n t e d in water. Growth kB o|o Maximum G odronia 1 8 12 20 25 30 35 TEMPERATURE (C °) i*ig. 9* Percent maximum growth ol“ Phomopais vaccinil and Godronia caaaandrae f . vaccinli at varioua temp­ eratures* Each point on the curves represents average diameters or 5 colonies of 2 separate iso* lates of each fungus, Measurements were taken after ll|. days growth-on £ PDA at the respective temperatures• k9 necrotic tissues by the lfjth day on some plants and con­ tinued to appear for 2-3 months (Pig. 1 2 - D ) . Extensive necrosis surrounding the inoculated branches did not develop on plants inoculated w i t h G o d r o n i a . The disease continued to develop for several months even when green­ house temperatures exceeded 27-30 C. D e a d tissues were sharply demarcated from living portions of inoculated stems (Pig. 1 2 - C ) . All lesions did not expand on Tr and 2-year-old stems (Pig. 11-E) and only 60% caused wilt. Those lesions which expanded on older stem3 did so very slowly, eventually killed most of the inoculated plant but (Pig. 1 1 - B ) . Five inoculated plants on which Phomopsis lesions did not develop, were exposed to moisture stress 1 year after they were inoculated. Cankers did not develop, even though Fhomopsis could still be isolated from the inocula­ tion sites. In another experiment, Godronia cankers which were apparently walled off, were reinoculated with P h o m o p s i s . The reinoculated branches died within 3 weeks. Control inoculations with blocks of hk PDA did not expand. The experiment was executed during the summer when temperatures in the greenhouse exceeded 30 C. These stems died more quickly than mo3t stems inoculated with Phomopsis alone. The possibility of Godronia and Phomopsis being synergistic warrants further study. $0 Pig. 10. Naturally occurring and artificially induced symp­ toms on blueberry stems. A) Callus 'ormation from strip of cambium left protruding into mechanically girdled portion of a 1-year-old stem. Note the similarity to the naturally occurring callus along the edges of a canker caused by Godronia (B). B) Naturally occurring callus along edges of a canker caused by God r o n i a . C) Gray centered lesions and maroon colored epidermis after a plant was inoculated with Godronia and grown for the summer and f o l ­ lowing winter in a cold frame. D) Large canker on the crown of a dead 3-yearold plant inoculated with Godronia via a chip wound. The plant was alive and uncankered prior to exposure to winter conditions in a cold frame. Note the pycnidia below the chip wound. 51 52 Pig. 11. Results of artificial inoculations. A) Lesion on Jersey variety plant 11 weeks after inoculating wit h G. cassandrae f . sp ira eic ola . B) Control plant (a) and plant inoculated with ■ £.* vaccinii (b) several months after inocula­ tion. C) Canker on 2-year-old Jersey variety stem 6 months after inoculation with P. vaccinii in the field during May. Note that the canker is long and narrow. D) Lesion on stem of Jersey variety plant 11 weeks after inoculation w i t h G. cassandrae f . vaccinii from \T. ang ust ifo Tiu m. Note the pycnidia on the lesion. E) Lesion on Bluecrop variety plant 6 months after inoculation with P. vaccinii in the greenhouse. Note the similarity to lesions caused by cassandrae f . vaccinii (D). 53 514 - Pig. 12. Plants artificially and naturally infected w i t h £• vaccinii . A) Natural infection on i^-year-old stem of W e y ­ m o u t h variety. Note the flaked bar k and the pycnidia w h i c h occur around the entire stem. The stem was dead when the photogra ph was taken in August. B) Long, narrow canker on l4.-year-old Jersey variety stem inoculated in the field w i t h P_. v a c c i n i i . The stem was inoculated in May and was c a n ­ kered by July. C) Sharp demarcation between living and dead tissues on 1-year-old stem inoculated wit h P. vaccinii in the greenhouse. D) Pycnidia in necrotic tissue on 1-year-o ld stem inoculated w ith .P. vaccinii in the g r e e n ­ house . 11 • :I | i I; • t| U 1 j i 56 Both Godronia and Phomopais w ere reisolated from their respective inoculation sites for up to 1*5 years after inoculations. Both fungi could also be re^solated from inoculation sites which did not appear to be active and which did not have symptoms of canker. None of the plants inoculated with Phomopais or Godronia developed overgrowths or t u m o r s . Field studies: After it had been established that both Godronia and Phomopsis were pathogenic on blueberries in Michigan, 3 studies were designed to define further s ymp ­ toms caused by each organism in the field: a) development of symptoms was followed on naturally Infected plants tagged in the field; b) w ilt ed branches were collected from fields in various areas in Michigan and Indiana and were studied in detail; c) plants in the field .were i n o c u ­ lated with Godronia and Ph omo psi s. Studies of naturally infected p l a n t s ; Diseased blueberry plants were tagged in 3 fields during April, The fields and plants studied were as follows: lower Michigan 1968. a) a central (CLM) field with Earliblue variety plants infected w i t h both Phomopsis and G o d r o n i a ; b) a northern lower Michigan (NLM) field with Jersey variety plants infected by G o d r o n i a ; c) an upper Michigan (UMF) field with several unnamed varieties infected by G o d r o n i a . Data confirming that Phomopsis was the o n l y pathogen in some fields were not available until July, Therefore these fields were not included in this study., 57 Five randomly selected diseased plants and at least 5 branches/plant were tagged in each field during April. Additional stems were tagged as they wilted and as new shoots developed. Symptoms and occurrence of.new infection were recorded every i|_—6> weeks during the 1968 season. The identity of fungi causing disease was con­ firmed by isolating from samples of tissue collected at various times throughout the growing season. Observations of plants tagged in the ITMF and NLM fields: By the end of April, 1- and 2-year-old branches were covered w i t h lesions of various sizes (Fig. 1, 2). W h e n numbers of lesions on 1- and 2-year-old stems were counted on tagged branches and on a random sample of ninetythree 1- and 2-year-old stems collected from the 2 fields, an average of 20.9 lesions was f oun d on each stem and of all lesions w ere centered by a leaf scar. percentage of nodal lesions was greater w i th 6 7 .9# of all lesions being The in the NLM field, at leaf scars. Most lesions were less than 1.5 cm long, but larger lesions with pycnidia were also common B ) . In the UMF field, (Fig. 2-A, the bases of m a n y branches were "peppered” wi th numerous red spots 1 m m and less in diameter. Godronia was isolated from such incipient infection sites. ded in size, coalesced, The spots gradually expan­ and by late August-October, the epidermis of the bases of the stems ha d turned red-brown, was flaking and splitting (Fig. 6-A, D, C), and as indicated 58 by isolations, thoroughly invaded by G o d r o n i a , M a n y of the larger internodal lesions expanded, but appeared to become walled-off and localized by midsummer (Pig. 3 -C) • It was not determined whether these cankers expanded d u r ­ ing the winter and spring. On stems which were more than 2-years-old, cankers were often 5 - 1 5 cm long and caused stems to appear gnarled, flattened or depressed (Pig. 3-A, B). The bark on f l a t ­ tened and depressed areas was usually flaked and cracked (Pig. 6-C). In wide, deep cankers,the xylem was exposed and the cankers often girdled more than 3 / k cumference of the stem (Fig. 3 - B ) . on such cankers. of the cir­ Pycnidia were not found X y l e m discoloration often occurred in parts of the stem w hic h were not cankered externally or in areas where only small lesions were visible. Flaking and splitting of the bark was common on such stems. ronia was isolated f r o m flaking, splitting, God­ or symptomless bark. Stems on which buds were beginning to expand wilted as early as May in b oth NLM and UMF fields. field, In the UMF leafed-out branches began to wilt during mid-June and continued to wilt until August (Fig. II4., 1J?-A, C) . Wi lting in the NLM field started later in June and con­ tinued until September. In both fields, 1 of the stems wilted because Godronia grew Into the bases of stems from infected crowns. Some stems wilted without visible cankers 59 whereas others did not wilt even when they were completely girdled by cankers exceeding 5 cm in length. Pycnidia were produced on lesions beginning in March-April in b o t h fields (Pig. 1-C, 2-A, B). Occas- sionally, pycnidia appeared in fissures in the bark and were not confined to lesioned areas (Pig. 6 - B ) . Apothecia (Pig. 16) were abundant on pruning stubs, along the length of erect, dead stems and on dead twigs accumulated around the crowns of plants. Apothecia occurred only on dead wood, and occassionally were found in dead tissues on unilaterally-killed stems. A few n e w lesions were observed in late July. Pycnidia did not appear on the lesions and lesions did not enlarge. On October 9, small darkened zones resem­ bling water soaking were observed along internodes of stems formed during the 1968 season. Isolations showed that G o d ­ ronia was present in these tissues. Small red spots (Pig. 1-A), often centered by small pockets of necrosis were observed on the stems by December 3* Isolations of God­ ronia from these tissues suggested that the red spots were incipient lesions caused by Godronia. Godronia was also isolated from reddened tissues surrounding leaf scars in December. Infection of stems formed during the 1968 growing season apparently had occurred before October 9* but symp­ toms were not apparent as lesions until December. Infection periods will be discussed further in Part II of this thesis. 60 Observations of plants tagged in the CLM fi e l d : In the CLM field in which Earliblue plants were infected by b o t h Godronia and P h o m o p s i s , 3tnall lesions were not as common as in the other fields* sent on older Large cankers p r e ­ (3 to 5-year-old) branches often extended into the crown. Both Godronia and Phomopais could be i so­ lated from such cankers, Phomopsis infections usually started on h ig h e r parts of the stem and gradually p r o ­ gressed down the cane, Frequently, eventually killing the entire branch. it was impossible to tell whether Phomopsis or Godronia killed the infected stem since both could be iso­ lated from the diseased tissue. Phomopsis was also found in the crowns of plants and appeared to be actively involved in killing branches originating from the crown. It was not possible to determine whether the crown was infected directly or via infected branches leading to the crown. Cankers from which only Phomopsis was isolated tended to be long in relation to their width and were usually cov­ ered by unbroken bark or epidermis (Fig. 11-C, E; 1 2 - B ) . On these canes infected by Phomopsis but without cankers, the bark and/or epidermis were commonly flaked and split­ ting (Fig. 1 2 - A ) . During April and May, dead tips were noted on many stems on which buds were beginning to expand. All of the stems were growing from the crowns of established plants and had been formed late during the 1967 season. Symptoms 61 resembled those caused by cold injury (614.), Phomopais twig blight (68), and Botrytia twig blight (6l|., 70). Usually 10-30 cm of the tips were dead and living tissues were sharply demarcated fr om the dead tips. The pith of living portions of the stems was chambered and brown for several cm below the killed tissues. In order to determine whether pathogenic fungi were associated with these symptoms, Jersey, Pemberton, 102 and Blueray variety stems with dead tips were collected from several fields. One cm long sections were cut fr om the juncture of living and dead tissues and placed on % PDA. The fungi identified growing from such tissues are listed in Table 8. Several other fungi includ­ ing Cytospora s p ., Dendrophoma s p ., Alternaria s p p ., and Epicoccum s p . were also commonly found in such tissues. Table 8. Fungi associated w i t h symptoms resembling cold injury, Phomopsis and Botrytis twig blights. No. sections sampled 102 Godronia 8 No. sections with fungi Phomopsis Botrytis Coryneum 2k 3 16 One to $-year-old stems did not wilt in the CLM field until July-August. Phomopsis pycnidia appeared d u r ­ ing August-October and were most prevalent later In the season on older, dead stems and occurred along the length of the dead branches (Fig. 1 2 - A ) . No perithecia of 62 Diaporthe were found in the CLM field or in any other field in Michigan and Indiana during the course of this research. Godronia pycnidia appeared during March-April as in the N LM and UMF fields. Godronia apothecia were found on pruning stub3 and some dead stems, but were not as common as in the other 2 fields. Survey of wilted b r a n c h e s : A detailed survey of wilting branches in Michigan and Indiana was conducted. The goals of this study were to: a) further define the symptoms caused by each organism; b) determine the rela­ tive severity and distributions of the 2 diseases in the Great Lakes blueberry region. Random samples of wilted stems were collected and the variety, location in the state, age of sampled stem, symptoms on the stem, degree of wilt, and where the stems originated on the plant were recorded for each branch. Earlier observations indicated that xylem discoloration (XD) was common in the stems of wilting stems. Each stem was cut into 1-2 cm long sections and the presence, tion, and extent of XD were recorded. loca­ At least 3 sections, taken from parts of the stems with canker symptoms or XD, were placed on PDA to isolate fungi in the tissues. The number of plants infected in each field from which wilted branches were collected was recorded in order to assess the relative severity of the 2 diseases. 63 Pig. 13. Artificially induced Qodronia lesions. A) Lesions w h i c h developed on a 6-mont h-o ld stem which was kept in a cold room f or 6 months. The stem was collected f r o m the field in October and showed only small pockets of water soaking in the cortex and reddening of leaf petioles at that time. B) One-year-old stem inoculated with and killed by Go dr o n i a in the field. The stem was inoculated in September, 1967 > and w i l t e d as buds expanded d u r i n g late April, 1968. C) Lesions on a 1-year-old stem inoculated with Godronia in the field during April. Photograph w as taken in early June. 64 B \ 65 Pig. l!j.. Typical flagging seen in fields in w h i c h either Phomopsis or God ron ia wer e epiphytotic. Godronia was epiphytotic in this upper Michigan field. The photograph was taken in July. 66 67 Fig. 1£. W i l t e d bra nches and di sco lor ed leaves. A ll stems w e r e inf ected w i t h Godronia and were phtographed in July-August. W i l t e d branches and discolo red leaves caused by Phomopsis were indistinguishable f r o m those shown here. A) Typical "flag11 or wilted branch. B) D isc ol o r e d leaves. The first leaf at the left in each row is unaffected. The various shades of red and brown were typical. C) W ilt ed leaves on a single branch. D) Ma rg i n a l b rowning of leaves of affected branch. The ma rg ins of leaves often showed the earliest signs of wilt. Leaf at the far left is u n a f ­ fected. * E) Premature reddening of leaves on affected stem. 68 69 Pig. 16. Apothecia of (x. casaandrae f . v a c c i n i i . A) Apothecia on dead 2 to 3-year-old stem c o l ­ lected in late July (3 X). B) Lateral view of apothecia C) Open apothecia after being placed in a m o i s ­ ture chamber for 2\\. hours (50 X). (25 X). 70 71 Leaves on stems infected with either Qodronia or Phomopsis often turned red or yellow before they wilted (Table 9, Fig. 15-B, D, C ) . Leaves on stems infected with Phomopsis showed this characteristic more often. Margins of leaves on affected branches often darkened or wilted before the remainder or the leaf (Fig. 15-A, D ) . Branches killed by Phomopsis were older than those killed by Qod­ ronia (Table 10). Most stems killed by Phomopsis were 3- years-old, whereas most stems killed by Qodronia were 2years-old (Fig. 17). Both fungi were associated with each of the symptom categories observed on wilted stems (Table 11 ). The distribution of symptoms (Fig. 18) on sections cut from wilted stems collected in fields in which Phomopsis was the only pathogen isolated (PH-Fields) differed from the distribution of symptoms on similar sections from fields in which Qodronia was the- only pathogen found (GDF i e l d s ). The lesion - young canker and callused canker categories were more frequent in GD-Fields, whereas branches which were wilted, but otherwise normal in appearance occur­ red more frequently in PH-Fields. Both fungi were associa­ ted with each symptom category in the respective fields (Table 12). Qualitative differences within symptom categories were present in these comparisons. Cankers found in PH- Fields tended to be long and narrow, and were covered with 72 Table 9« Characteristics of leaves on branches killed by Godronia or Phomopsis.a Fungus red Phomopsis Godronia No. stems with discolored leaves wilted, brown, yellow red-yellow or defoliated 17 9 7 157 k k 2 137 a Data based on 326 wilted branches collected from 19 fields during the 1968 growing season. Table 10. Average ages of wilted branches from which God* ronia and Phomopsis were isolated. Fungus Isolated Average age (years) Total number wilted branches sampled Godronia 2.38 128 Phomopsis 2.96 181*. Both 2.67 21 73 Table 11. Association of Godronia and Phomopais with symptoms observed on wilted stems." Percent sections with fungus Symptom category Godronia Phomopsis None 25.6 63.1 Lesion-young canker 66.lj. 20.6 Developed canker 32.5 52.8 Callused canker 61.5 27.2 Discolored bark or epidermis 33.2 1*8.5 Split or flaking bark or epidermis 39.3 k5-k a Based on 1696 sections from 314-7 flags collected during the 1968 season. 7k unbroken bark or epidermis (Pig. 12-B). Cankers in GD- Fields were wide in relation to their length and often completely girdled the infected branches. Xylem was exposed due to distinegration of the bark and epidermis over the cankers (Pig. 3"B). Xylem discoloration occurred at some point in the xylem of wilted branches regardless of the age of the stem or the fungus isolated. Zones of XD varied in length from those shorter than 2 cm to others which extended the length of the stem. Zones of XD were usually 2-l|. cm long at the onset of wilt symptoms and were longest on stems with browned leaves. There was no correlation between the pathogen iso­ lated from the xylem and the extent or position of XD. Infected stems wilted above regions of XD and leaves below remained healthy. Wilting was confined to 1 side of 3 branches in which XD was unilateral. Both fungi were associated with all of the symptom categories occurring in areas of XD (Table 13). It i3 especially interesting to note that many stems infected with Godronia were not cankered or lesioned in areas of XD. Phomopsis and Godronia were both widely distributed in Michigan (Pig. 19). However, in fields north of Mason County, Godronia was epiphytotic and Phomopsis was not found. In the center of the "blueberry belt," both fungi were common. 75 mi G odronia w * r Phomopsis o Both No. Wilted Stems 75 30 3 Age (y e a rs ) Pig. 17• Distribution of ages of wilted stems from which Godronia, Phomopsis, or both fungi were isolated. Data are based on 3*17 wilted stems collected from 18 fields during the 1968 season. Identi­ fication of fungi based on isolations made from discolored xylem in each stem. 76 Table 12. Association of Qodronia and Phomopsis with symptoms on sections from stems collected in fields in which either Qodronia (GD-Pields) or Phomopsis (PH-Pields) was the only pathogen isolated.*1 PH-Pields QD-Pields Symptom Percent Total sections sections with Godronia observed Percent sec­ tions infected with Phomopsis Total sections observed None 96.3 27 38.6 U4 Lesion-young canker 97.0 66 78.6 14 Developed canker 89.1 101 73.3 1$0 Callused canker 95-9 lj-9 69.0 29 Discolored bark or epidermis 87*3 79 76. k 110 Split or flaking bark or epidermis 94.1 20h 73-0 159 a Godronia data based on $ 2 6 sections from $ northern Michigan fields; Phomopsis data based on ij.96 sections from 7 Indiana and southern lower Michigan fields. 77 Phomopsis Fields D Godronia Fields ■ 200 A Pig. 18. B C D E F Sym ptom C ategory Distributions of symptoms in fields in which either Godronia (Godronia Fields) or Phomopsis (Phomopsis Fields) was the only pathogen iso­ lated. Godronia data are based on-f>26 sections from 5 northern Michigan fields." Phomopais data are based on lj.96 sections from 7 Indiana and southern lower Michigan fields. Symptom cate­ gories are a3 follows: A = none; B = callu3ed cankers; C = lesion-young cankers; D = discolored bark or epidermis; E = developed cankers, F = split or flaking bark or epidermis. 78 Table 13. Association of Phomopsis and Godronia with symptoms observed on sections of stems with discolored x y l e m a Symptoms Percent Sections with fungi Godronia Phomopsis Total sections None 22.7 71.0 66 Lesion-young canker 70.9 2$.8 31 Developed canker lj.0.6 S7.2 138 Callused canker 61.7 350 3k Discolored bark or epidermis 31*. 2 60.5 .76 Split or flaking bark or epidermis 39.14- 55.8 231 a Based on sections taken from 3U-7 wilted stems during the 1968 growing season. 79 • s FtokU w ith Oodronki S Fields w ith Mtomopsis NUM MMIIM Pig. 19.' Distribution of Phomopais and Godronla in Michigan and Indiana blueberry fields. Based on samples collected from 57 fields during 1965-1968. Godronia was epiphytotic north of Oceana County and Phomopsia was epiphytotic in several Indiana and Van Buren County fields. 80 Both fungi attacked all major varieties of blue­ berries grown in Michigan (Tables llj., 15)» but the If. most important varieties appeared to be most susceptible to Godronia (Table 16). Godronia is probably the more impor­ tant pathogen in Michigan. Jersey variety appeared to be most susceptible to Godronia canker and Hancocas "most resistant. Earliblue was very susceptible to Phomopais and infected plants were often non-productive. Inoculation of plants in the field: r * Symptom pro- * gression was followed on plants inoculated with Godronia and Phomopais in the field. Beginning in April, 1968, 8- year-old Jersey variety plants growing in the Michigan State University Orchard were inoculated every 2 weeks with Godronia and Phomopais. One plant was inoculated 10 times with Godronia and another inoculated similarly with Phomopais on each inoculation date. The same isolate of each fungus was used for all inoculations. Symptom p r o ­ gression was observed through the growing season. Both Phomopais and Godronia readily infected plants inoculated during April-June (Table lij.), but the percentage of inoculations causing disease decreased during July. Fur­ ther expansion of successful inoculations also stopped dur­ ing this period. It was not determined whether cankers expanded during the fall and winter. During the spring and early summer, Phomopais and Godronia lesions developed on 1 and 2-year-old stems (Fig. 13-C). A single 1-year-old 81 Table llj.. Blueberry variety Association of Godronia and Phomopais with varieties of blueberries grown in Michigan and Indianaa Percent 15&7 acreage0 -•Total number :fields Sampled Godronia ^homopsis present present Jersey 57.0 22 18 7 Rubel 11*. 30 11 5 8 Bluecrop 6.65 3 2 1 Earliblue 3-70 8 3 6 Stanley 3.06 k 2 1 Pemberton 2.75 3 1 1 Blueray 2.50 1 0 0 Berkley 2.00 1 0 1 Rancocas 1.30 1 1 0 Weymouth 0.83 6 3 k Collins 0.60 2 1 0 Dixie <0.92 1 0 1 Grover <0.92 1 0 1 Unnamed <0.92 k k 0 a Based on samples collected from 57 Michigan and Indiana fields during 1965-68. 0 Percentages based on data compiled by the Michigan Bl u e ­ berry Growers Association. 82 Table 15. Blueberry Variety Association of Godronia and Phomopais with varieties of blueberry grown in Michigan. % sections infected Godronia Phomopais No. observations Sections Fields Jersey 1*7-7 9.3 869 22 Rubel 38.0 1*2 . 7 1*91 11 3 . 1* 51.8 525 6 285 1* Weymouth 0 Unnamed 9 5 - 1* Earliblue 23.6 1*5-2 550 8 Bluecrop 66.7 1**3 253 3 Rancocas 91.3 0 1*6 1 16.6 1*8 3 Pemberton 6.2 Dixi 0 100.0 3 1 Berkley 0 62.£ 8 1 Collins 7.1 0 11* 2 Blueray 0 0 21* 1 Stanley 3.6 2.7 217 1* 68.9 71* 1 Grover 0 83 Table 16. Relative susceptibility if major blueberry varieties to Godronia and Phomopais,a Variety Field Susceptibility13 Godronia Phomopsis Jersey +++ + Rubel +++ ++ Bluecrop +++ + Earliblue +++ +++ a All plantp were naturally infecte i. k +++ = most severly affected; + = least severe. branch inoculated with Phomopsia in April, died by mid-May. One cane inoculated with Godronia in September, 1967, died as buds began to expand in April (Pig. 13-B). Pycnidia developed on Godronia lesions in about ij. weeks after April, May and June inoculations, but not at all on 3terns inocu­ lated later. Pycnidia developed by March on the stem inocu­ lated in September, 1967. Pew pycnidia developed on stems inoculated with Phomopsia. Slow spreading cankers developed on 3- to 5 - y e a r o l d stems when chip or pin prick wounds were inoculated with Godronia or Phomopsia. Phomopsia cankers tended to be long, narrow and covered by unbroken bark or epidermis (Pig. 11-C, 12-B}. No overgrowths developed on any of the inoculated plants. The decrease in percentage of inoculations causing disease corresponded with an increase in daily temperatures. It is interesting to note that inoculations with Phomopsia in July caused cankers, but those made with Godronia did not. That Phomopais grows better at higher temperatures (Pig. 9) may be an explanation of this observation. Low incidence of wilt on inoculated plants: Cankers readily developed on plants inoculated with G. caasandrae f . vaccinii in the greenhouse, but few stems wilted. Examina­ tion of wilted stems collected from the field had shown: 1) xylem discoloration occurred at some point along all wilted stems; 2) leaves were always wilted above XD and 85 Table 17. Results of inoculations made on Jersey variety plants in the field with Qodronia and Phomopais.5 Date of inoculation September 25, 1967b % inoculations causing, cankers Qodronia Phomopais 100 — 90 90 100 60 May 18, 1968 80 20 May 31, 1968 90 ko June 19, 1968 10 80 July 1, 1968 0 50 July 15, 1968 0 20 July 29, 1968 0 0 60 30 April 21’ , 1968° May 3, 1968 August 27, 1968 One plant was inoculated 10 times with Godronia and another 10 times with Phomopsia on each date. None o f the controls developed cankers. b One stem wilted during April, 1968. c One stem inoculated with Phomopais wilted during May, 1968. 86 healthy below; 3) Godronia was present in XD tissues; i;) many uncankered stems wilted; 5 ) many severely cankered stems did not wilt. It seemed probable that wilting occur­ red only when the fungus was able to invade xylem tissues. Several observations suggested that low tempera­ tures and/or dormancy predisposed stems infected with Qod­ ronia to wilt. Stems inoculated with Godronia wilted when greenhouse temperatures were maintained lower than 18 C, whereas lesions appeared to become walled-off when green­ house temperatures were 25-30 C. Similarly, observations of naturally infected plants indicated that many small lesions observed in April were localized during the warmer summer months. When plants were inoculated with Godronia in the field (Table 17), only spring and fall inoculations caused cankers and the only stem to wilt was inoculated in September. 37, 73). Similar data had been reported by others (35, Experiments of others (16, 37, 52) and data pr e ­ sented above (Pig. 9) showed that G. cassandrae grew well at O-I4. C and that the organism did not grow at temperatures above 30 C. Exposure to $ - 6 C for 6$0 hours satisfied the cold requirement of blueberries (32). Godronia, then is able to grow well at temperatures inducing dormancy of blue­ berries. The following experiments were designed to deter­ mine whether low temperatures and/or dormancy predispose V, corymboaum plants infected with (J. cassandrae to wilt. 87 Effect of dormancy on localized lesions: The objec­ tive of these experiments was to determine whether appar­ ently localized lesions would expand when plants were dor­ mant. Five 1-year-old plants and two 3-year-old plants were inoculated with Godronia during March, grown in a 1830 C greenhouse, and transferred to a cold frame in June. Plants were returned to the greenhouse the following March. Controls consisted of inoculated plants grown in the green­ house and noninoculated plants grown in the cold frame for the duration of the experiment. Lesions had the following characteristics when plants were transferred to the cold frame in June. Centers of lesions were necrotic and stem tissues were raised along the edges of necrosis. Epidermis was raised and was red- brown to maroon colored several mm in advance of necrosis. Lesions appeared to be walled off. Centers of lesions turned gray on plants grown in the cold frame. Maroon discoloration of epidermis was more extensive on plants grown in the cold frame than on plants grown in the greenhouse. (Fig. 10-C) None of the 1-year- old plants wilted when they were returned to the greenhouse in March. One 3-year-old plant inoculated via a chip wound in the crown severly cankered and died before leaves were fully expanded (Fig. 10-D). 88 In another experiment three 1-year-old plants were inoculated and grown in the greenhouse as above and were transferred in June to a 2 C coldroom for 2.5 months. Con­ trols consisted of inoculated plants grown in the green­ house and noninoculated plants placed in the cold room. Lesions did not expand and stems did not wilt on any of the plants in this experiment. Effect of temperature on lesion development: The objective of this experiment was to determine whether low temperatures favored initiation of infection and early lesion development. Ten inoculations were made with the same isolate of G. cassandrae f. vaccinii on each of 15 plants. Each inoculation site was pierced a single time with a fine needle. Immediately following inoculation, 5 plants were placed in 5 or 15 C growth chambers (R. H. = 70-90#; light = 18 hour photoperiod, 500 fc flourescent source), or in a 18 C mist chamber (R. H. = 90#; light = normal daylight supplemented by 2 hour 200 fc incandes­ cent source). Lesions were initiated at each temperature. expanded most rapidly at 18 C (Table 18). Lesions Fifteen C was more favorable than 5 C for lesion expansion and pycnidia development (Table 18). Another experiment was performed to determine whether a previous 10 day incubation period in high humidity at 18 C would increase the rate of lesion expansion at 5 and 15 C. 89 Two plants wera inoculated and incubated for 10 days at 18 C in the mist chamber. Following incubation 1 plant was placed at 5 C and the other at 15 C. Lesion expansion (Table 19) was similar to that (Table 18) on plants grown at 5 and 15 C without previous incubation at 18 C. No stems wilted after 27 days exposure to 5 and 15 C . An experiment was performed to determine whether lesions on plants grown for 37 days at 5 and 15 C would continue to expand at low temperatures; and become local­ ized at high temperatures. Plants grown at 5 and 15 C for 37 days were transferred to 2, 8, 10, 20, and 30 C growth chambers. One plant from 5 and another from the 15 C cham­ ber was placed into each growth chamber. Plants were grown at these temperatures for 97 days and were then transferred for 60 days to a 20 C chamber. Lesion size was inversely related to temperature (Fig. 20). Lower temperatures favored lesion development. Lesions appeared to be walled off at 20 and 30 C. Plants grown at 2 and 8 0 grew rapidly when placed at 20 C indi­ cating that they had been dormant. Stems did not wilt on any plants in this experiment. This experiment was similar to the previous experi­ ment except that plants were inoculated, incubated in the 18 C mist chamber for 12 days, and ware placed directly into 2, 8, 10, 20, and 30 C growth chambers. Three plants, 90 Table 18. Expansion of lesions and formation of pycnidia on plants inoculated with Qodronia grown at 5# 15, and 18 C. Average Rate Exposure No. lesion No. lesions period lesions length (days) counted lesions expansion with pycnidia (mm/day) (mm) Temperature (° c) 5 27 50 2.1* .09 0 5 37 50 3-0 .08 3 15 27 50 3.2 .12 11* 15 37 50 k-3 .12 15 18 12 121 3*2 CVJ • t*- 0 18 18 50 3-7 .21 0 Table 19* Expansion of lesions on plants inoculated with Godronia and grown for 10 days in a high humidity c h a m b e r a t 18 C and then for 23 days at 5 and 15 C. Temperature (° C) Number lesions measured Average length lesions (mm) Rate lesion expansion (mm/day) 5 10 2.3 .08 15 10 5.2 .16 Lesion size (cm) 91 Temperature (C) Pig. 20. Pinal average size of lesions caused by Godronia on inoculated 1-year-old Jersey variety plants after exposure to various temperatures. Plants were exposed to $ (CD ) or 1 $ (OV ) C for 37 days, followed by exposure to 2-8, 10, 20, or 30 C for 97 days, and 60 days at 20 C. Data for each bar are based on average length of 10 lesions on 1 inoculated plant. 92 each inoculated 10 times were placed into each chamber. Plants were grown for 122 days at the respective tempera­ tures and then grown for 63 days at 20 C. Necrosis was most extensive (Pig. 21) at 2 and 8 C. At these temperatures, lesions were covered with pycnidia, coalesced, and extended the length of the inoculated stems. Five of 12 inoculated stems wilted when plants from 2 and 8 C were grown at 20 C. Rapid growth of these plants at 20 C indicated that they had been dormant. No differences in symptom development were noted between plants exposed to 2 and 8 0. Plants exposed to 10 C did not grow rapidly when placed in the 20 C growth chamber and stems on these plants did not wilt. Lesions appeared to be walled-off at 20 and 30 C. Data from this series of experiments suggest that initial infection and lesion development is related to growth of Godronia at various temperatures, but that later lesion expansion is inversely related to temperature. Dor­ mancy of host plants favored fungal invasion of xylem tis­ sues and subsequent development of blight symptoms. The inability to control light and relative humidity detracts from the value of these data. However, further experiments tinder more controlled conditions are certainly warranted. Callused cankersi Several different types of over­ growths were commonly associated with cankered blueberry stems. Detailed observations in the field and laboratory Lesion size (cm) 93 Temperature ( C) Pig. 21. Pinal average size of lesions caused by Godronia on inoculated 1-year-old Jersey variety plants after exposure to various temperatures. Plants were exposed to 18 C for 12 days, followed by 122 days at 2-8, 10, 20, or 30 C, and 63 days at 20 C. Data for each bar are based on average length of 30 lesions on 3 inoculated plants. Five of 12 inoculated 3 tems wilted at 2-8 C. 914 - showed that overgrowth symptoms could be divided into 5 categories based on the texture of the callus and the position of calluses on the affected plant. were as follows: 8 -A calluses These calluses (Pig. I4.-A, B) were calluses occurring at the bases of lateral branches; 8 -B calluses (Pig. i*-C, D) were corky calluses extending the length of affected stems; 8 -C calluses (Pig. 5-A, B) were corky and located along edges of cankers in the crowns of affected plants; 8 -D calluses (Pig. 5-D, E; 10-B) were corky cal­ luses occurring at edges of cankers located along the affected stem; 8 -E calluses (Pig. 3-C, D) resembled healed wounds. The following data are based on field and laboratory examination of over period of 3 years. 0 callused stems collected over a Regardless of the type of overgrowth, all calluses were associated with cankers. found only on stems which were cankered. Calluses were Neither boring insects such as the dogwood borer (Thamnosphecia scitula Harris), nor signs of their activity were observed in cal­ lused tissues. Nematodes were commonly found in 8 -B cal­ luses, but no attempt was made to identify or culture these organisms. No calluses were found on the excavated roots of 11 plants with callus symptoms on stems. Callus tissue was confined to the cambium, phloem, and cortex and could be easily removed from the xylem. 95 Stems of plants with callus symptoms often wilted and died. The xylem of dying stems was discolored at some point along the length of the branch. Since all of these stems were also cankered, it wa3 impossible to determine whether the wilting was associated with the organism caus­ ing the canker or the agent causing the callus. Frequently, no canker fungi could be isolated from cankers on callused stems, and examination of the growth ring initially affected indicated that cankers were often initiated 3-5 years before the stem callused and wilted. The leaves of callused branches usually turned yellow or red before they wilted. Often, calluses concealed by dense foliage could be loca­ ted by tracing along branches with discolored leaves. . ‘ S ymptoms 8-C, 8-D and 8-E could always be found in fields in which Phomopais and/or Godronia were common; however, these ^symptoms were more pronounced and more com­ mon in GD-Fields. Symptoms 8-A and 8-B were not found in all fields in which Godronia or Phomopais were found, but Godronia was present in each of the fields in which these symptoms were found. Pruning of affected branches seemed to effectively check the spread of these symptoms in affec­ ted fields. The association of fungi and bacteria with callused canker categories is shown in Table 20. These data are based only on those callused cankers which were sectioned and planted as indicated in Methods and Materials. 96 Table 20. Organisms associated with callused cankers. Fungus 8 -A 8 -B % % Callus type 8 -C 8 -D % % 6 -e % 21.2 51.8 57-9 82.U 3U-.5 3.8 1.8 0 25.9 37.1 Coryneum 11.5 0 0 1.9 0.9 Fusarium 1.9 19.6 0 0.9 0.9 Alternaria 23.1 30.8 19.3 50.0 5^.3 Epicoccum 36.5 1.8 10.5 28.7 W-B Botrytis 7.7 0 5.3 5.6 It. 3 Papulospora 1.9 12.5 3-5 5.6 0.9 Bacteria 2.0 lj.0.0 3.0 5.0 5.0 52 56 57 108 116 Godronia Phpmopsis Total sections Qodronia was commonly isolated from each type of callus. Categories 8 -D and 8 -E were also frequently inhabi­ ted by Phomopais. Coryneum and Fusarium were observed most often on 8 -A and 8 -B, respectively. Bacteria resembling A. tumefaciens were common in 8 -B calluses, A series of experiments designed to determine the cause of callusing produced mostly negative results. When­ ever actively growing plants were inoculated via chip wounds, 8 -E calluses developed. in appearance, Since control wounds were similar it was concluded that 8 -E calluses were a 97 wound response and probably develop whenever slowly develop­ ing cankers occur on rapidly growing stems. Although 8-D calluses did not develop on plants inoculated with Godronia and Phomopsia, the symptom was induced by mechanically girdling rapidly growing stems (Pig. 10-A). These results suggest that 8-D calluses result from the girdling action of canker fungi on rapidly growing stems. Circumstantial evidence indicated that cankers caused by Godronia and Phomopsia play a role in 8-D callus formation. Both fungi were commonly associated with this symptom (Table 20). The symptom was very common in upper Michigan fields in which Godronia was the only pathogen found. Histological observations of 8-D calluses from which Godronia was isolated showed that the tissues were permeated with hyphae (Pig. 30-A). In short, when conditions are favorable, 8-D calluses probably develop in response to the slow girdling action of cankers caused by Godronia and to a lesser extent by Phomopsia which grows more rapidly in infected tissues. Whether 8-A and 8-C calluses fall into this category is unknown. Godronia, however, was commonly associated with these tissues (Table 20). The cause of 8-B calluses was not determined. The symptoms were similar to symptoms of crown gall in British Columbia (Pig. ij.-E). Attempts to transmit the symptom by using small pieces of callus tissue to inoculate blueberry plants failed. Similarly, all bacteria isolated from 8-B 98 calluses failed to induce symptoms on inoculated blueber­ ries and several herbaceous indicators of crown gall. Control plants inoculated with known A. tumefaciens also failed to produce g a l l s . Even though there was evidence that the known cultures of A. tumefaciens had lost patho­ genicity* it was impossible to draw conclusions from the data. In summary, 8-E and 8-D calluses probably result from the girdling action of cankers caused by Godronia and Phomopsia. It is possible that 8-A and 8-C calluses are similarly caused, but proof is lacking. calluses is unknown. The cause of 8-B The callus syndrome on blueberries in Michigan merits further research. PART II. DISEASE CYCLE AND INFECTION STUDIES. Introduction: The underlying objective of this research was to obtain sufficient information to provide a basis for designing fungicide evaluation experiments. Attempts to control "Fusicoccum" canker with fungicides had been ineffective (1 6 , 3 1 » 3 7 * 7 3 )• It seemed likely that such failures were due to improper timing of fungi­ cide applications. These studies were designed to deter­ mine when and how Godronia infects blueberries in Michigan. Methods and Materials. Availability of inoculum: Observations of rela­ tive numbers of apothecia and pycnidia present in blueberry fields were recorded at various times during the 1965-1968 growing seasons. In addition, fruiting bodies were col­ lected from several different Michigan fields at various times during 1965-1968. Fruiting bodies were placed in moisture chambers at 20-25 C for 2l\. hours and examined microscopically for the presence of spores. Other inoculum sources: Groves (28) reported in 1965 that (}. cassandrae probably existed in several forms which were found on several genera of plants. Populations of plant species known to be hosts of forms of Godronia were surveyed in areas adjacent to blueberry fields in which 99 100 Godronia was epiphytotic. Isolates from these hosts were used to inoculate healthy Jersey variety blueberry plants. Control inoculations were made with isolates of G. cassan­ drae from V. corymbosum. Infection periods; Healthy plants were exposed to natural inoculum in fields in which Godronia was known to be epiphytotic. Each month during the 1967 growing season ten 3-year-old and six l-year*old Jersey variety plants were placed in a single field, left for 1 month, and then replaced with a similar set of plants. Control plants were kept in a nearby noninfested field for the duration of the experiment. Counts of lesions were made periodi­ cally on all plants. Every 6 weeks during the 1968 season ten 1-yearold Jersey variety plants were placed into each of 2 fields (Field A was located in upper Michigan and Field B in north­ ern lower Michigan) each heavily infested with Godronia. Following each 6 week exposure period, plants were returned to a cold frame in East Lansing and replaced with a similar set of plants. Controls were plants kept in the cold frame for the duration of the experiment. All plants were cut into 0.5 cm long sections at the end of the experiment. A random sample of $ 0 sections was taken from each plant and placed on h PDA to determine relative levels of infec­ tion by Godronia. Relative levels of infection were expressed as the Infection Index. Infection Index was 101 defined as the number of sections yielding Godronia when sections from plants were placed on PDA. Thus the Infection Index would be 2 $ if Godronia grew from 2$ of the 500 sections taken from a given set of exposed plants. Experimental Results Availability of inoculum; Sporulating pycnidia were most abundant during Apr i l to mid-June (Table 2.) Conidia were observed washing down stems during April and May rains, but not during rainy periods of the remainder of the growing season. Although conidia were most abun­ dant during April to mid-June,some were present throughout the growing season. Pycnidia were found in all fields infested with Godronia. Apothecia were first observed in late April. Ascospores, however, were not mature until early mid-July (Table 22). Evidence of ascospore discharge, as indicated by empty asci, was noted by mid-July. Most ascospores had been discharged by mid-September, but some were present in asci in mid-October. New apothecia were formed by July on a stem from which apothecia were removed in April. Apothecia were not found in all Godronia infested fields, but were common in 8 fields in which Godronia was epiphy­ totic. Other inoculum sources; Apothecia and pycnidia of G. cassandrae were found on dead stems of V. angustlfolium and C_. calyculata adjacent to Michigan blueberry fields. Pycnidia were also found on dead stems of several species of Spiraea. Godronia was isolated from lesions and blighted stems of these h o 3 ts. Michigan isolates of <1. cassandrae 102 103 Table 21# Presence of Qodronia spores in fruting bodies collected at various times during the 1965-1968 growing seasons. Year® 1966 1965 Sample dates A c April 20-30 - - May M - June 1-15 - - June 16-30 - - July 1-15 - July 16-31 A 1968 1967 d A d A 0 * 4-4-4 4-4*4- - - - - •it ++4 4-4-4- * 4-4-4- * 4-4-4 - - - 4- 4-4-4- 4-4*4- + 4-4-4- - - 4-4-4* + 4-4-4- + - A ugust - M - + ++ + 4-4- S e p t e m b e r 1-15 - - + + 4- + - S e p t e m b e r 16-31 0 + - + - - - + - - - - 4- 4- - - - - - 0 4- October D e c e m b e r 1-3 - - mm a A = ascospores C = conidia + = few spores; 4-4- = moderate number spores; +++ - many spores. # = spores immature. 0 = no spores. - = no fruiting bodies sampled. • + 4- I 101* from V. angustifollum. V. corymboaum. C.. oalyculata. and Spiraea app.; and isolates from V. macrooarpon, V. angustlfolium. C . oalyculata. and Betula ap. obtained from other workers were studied. Isolates from £. oalyculata (Pig. 22-C, D) and V. macrooarpon were canary yellow to pale yellow in color, sporulated poorly, and produced abundant aerial mycelium on h PDA. Isolates from Spiraea spp. were similar except that colonies were pale yellow to tan in color (Pig. 22-C, D). Isolates from V. corymboaum varied from canary"yel­ low to brown or gray in color (Pig. 22-A, B, E, 23). Some isolates produced abundant aerial mycelium whereas others did not and were slimy in appearance on *§ PDA. ascospore isolates sectored occassionally. Single Cultural char­ acteristics were maintained when mycelial transplants were made. Sclerotia did not occur. Isolates from V. angustifolium (Pig. 22-A, B, C, D) were yellow to brown in color and sporulated well on *g PDA. Some isolates produced abundant aerial mycelium whereas others were slimy in appearance and produced little aerial mycelium. Porm beticola (Pig. 22-C, D) produced pale yellow to tan aerial mycelium on h PDA and did not sporulate. Growth of all forma of G. cassandrae (Table 22) at various temperatures was similar to growth of £. vaccinii 105 Table 22. Form of G. Cassandrae Growth of forms of Godronia cassandrae at various temperatures.a 0-2 Host Temperature (0) 12 20 8 25 30 35 Colony diameter (cm) cassandrae Chamaedaphnae 0.5 2.3 6.1j. 7.0 1*.8 1.2 0 vaccinii V. anfcustifolium 0.2 1.9 6.1 7.5 5-3 0.9 0 vaccinii V. corymbosum 0.7 2.1+ 6.3 7.1* 3.7 0.5 0 vaccinii V-. macrocarpon 0.5 2.3 7.2 3.6 1.0 0 beticola Betula sp. 0.2 0.1 2.3 6.5 1.0 0.1 0 spiraeicola Spiraea spp. 0.1+ 2.7 5.9 7.1* 5*5 1.2 0 a Growth of 5 Isolates grown on exposure to each temperature. PDA measured after 2 weeks Table 23- Spore morphology of forms of G. cassandrae found in Michigan.a No. spores Isolate measured Code Host Average size (*1) Range of size (M) % with % spores % spores 1 septum straight curved 30 SR-7 Spiraea 9.2 X 1-3 1 2 .7 -8.1 X 2 .3 -1.2 100.0 76.6 23.1* 50 SR-8 Spiraea 10.8 X 2.1 16.0-6.7 X 1.6-5.3 100.0 98.0 2.0 50 SR-1* Spiraea 9-8 X 2.0 12.6-6.9 X 3-2-1.3 100.0 80.0 20.0 30 CH-1 Chamaedaphnae 8.8 X 1.2 1 1 .5 -5-8 X 2 .3 -1.2 93.3b 100.0 50 CH-2 Chamaedaphnae 9.6 X 1 -1* 1 3 .8 -1*.6 X 1 .2 -1.5 7 8 .0b 90.0 10.0 50 Oct-2 V. angustifolium 9.3 X 1-7 13.8-3.5 X 2.7-0.9 8 6 .02 1*2.0 58.0 V. corymbosum unnamed var. 11.0 X 1-7 15.0-6.9 X 1 .6 -1.2 9 6 .6 b 1*0.0 60.0 50 50 50 50 50 No ’ed PC-11 AS-9 AS-10 AS -6 FC £ All All c Two 125 V. australe Jersey var. 12.2 X 1.6 1 6 .2 -8.2 X 2 .3 -1.2 100.0 68.0 22.0 V. corymbosum unnamed var. 10.0 X 1-5 17.3-7.5 X 2.1-1.3 100.0 1*2.0 58.0 V. corymbosum unnamed var. 10.0 X 1-3 1 2 .7 -8.1 X 2.3-0.9 9 8 .0b 18.0 82.0 V. corymbosum unnamed var. 9-8 X 1 -1* 13.8-5.8 X 2 .1 -1.3 9 6 .0b 21*.0 76.0 V. corymbosum Earliblue var. 11.2 X 1 .1* 15.i*-7.7 X 1 .0 -2.6 79.1° 91*. 0 6.0 spores measured in Ammon’s mounting modium (lactophenol) (2). other spores were non-septate. septate 12.8£; three septate 8.1$; number septations based on count of spores. 106 30 0 107 Pig. 22. Isolates of G. cassandrae grown on Jg PDA at various temperatures. A) Single ascospore isolates from corymbosum (a, b), single conidium isolates from V_. c o r y m ­ b o s u m (c, d), Michigan single ascospore isolate f r o m V_. anguatifollum (e), Nova Scotia isolate f r o m V. corymbosum (f) obtained f r o m C. L. Lockhart"^ Quebec isolate from V. angustifolium (g) obtained from E. Smerlis. All isolates g rown at 25 C. B) Same isolates as in (A) grown at 20 C. C) Quebec isolate of f_. beticola (a) obtained f r o m E. Smerlis, Quebec isolate of f. c a s s a n ­ drae (b) obtained from E. Smerlis, Michigan isolate of f. spiraeicola (c), M i c h i g a n i s o ­ late of f. cassandrae ( d ) . All isolates grown at 25 C. D) Same isolates as in (C) grown at 20 C. E) Response of single ascospore isolate from V,. c orymbosum to temperature. 108 109 Pig. 23. Variations in cultural m o r p hology of single a s c o ­ spore isolates of G. cassandrae f , vaccinii from V. c o r y m b o s u m . All are ij.-6 week old cultures on § PDA. 110 / Ill Pig. 21|.. Asci a n d spores of G. cassandrae f . v a c c i n i i . A) Asci mounted in lactophenol and stained w i t h 0 . 1 # cotton b l u e (800 X). B) Single ascospore m o u n t e d in lactophenol and sta i n e d with O.I 96 cotton blue (800 X). C) Ascus mounted in water D) Section cut through pycnidium on an infected stem (800 X ) . Cut w i t h an experimental m i c r o ­ tome (2 9 ) . (800 X). 113 Pig. 2£. Conidia of O. cassandrae (A, C, D, mounted in w a t e r and B in lactophenol and stained with 0.1% cotton blue). A) Conidia from p y c n i d i u m on Earliblue variety (800 X). Note the spore w i t h 3 septa. B) Conidia from pycnidia on Earliblue variety (2000 X ) . C) Conidia from pycnidium on Je r s e y variety (800 X ) . D) Conidia of f. spiraeicola (800 X). 114 115 from V. corymbosum (Pig. 9, 2 2 - E ) . Isolates from V. corym- bosum and V. angustifolium which produced viscous material in culture did so only at 20-25 C (Pig. 22-A, D). Ascospores of 0. cassandrae on corymbosum (Pig. 2I4.-B), V. angustifolium. and £. calyculata were 5-9 sep­ tate, straight, and measured 7 9 *1 -9 6 .14. x 1.0-1.3 M. (Pig. 2 I4.-A, C) measured 5*3-16.3 x 82.5-106.I4. ju. Asci Micro- conidia were observed only in pycnidia on dead stems of V. corymbosum. Microconidia germinated and produced typical cultures of G. cassandrae on PDA. Morphology of Conidia (Pig. 2I4.-D, 25) are summarized in Table 2 3 . Isolates of G. cassandrae from \f. angustifollum, Spiraea s p p .. and possibly (3. calyculata were pathogenic on V. corymbosum (Table 214.) . Pycnidia were produced on plants inoculated with isolates from V. corymbosum and V. angustIfolium, but not on cankers caused by isolates from Spiraea s p p . or calyculata. Godronia was reisolated from lesions developing from inoculation sites. Isolates from V. macrocarpon did not cause cankers on highbush blu e ­ berries, but epidermis turned tan around inoculation sites about 1 year after inoculation. No attempt was made to isolate G. cassandrae from such tissue. Infection periods; Plants introduced into the sin­ gle field in 1967 were not infected. It was further noted that in the spring of 1968 little new infection occurred in this field . During previous years 35-14-0 lesions/plant were 116 Table 2lj.. Pathogenicity of forms of G. cassandrae on Jersey variety blueberries. Source of isolate No. inocu­ lations % ‘inocu­ lations successful Form of G. cassandrae Host spiraeicola Spiraea spp. Michigan 1*8 cassandrae C. calyculata Michigan 52 0 cassandrae C. calyculata Q,uebeca 20 5.0 beticola Betula sp. Quebec® 20 0 vaccinii V. macrocarpon Wisconsin*3 21* 0 V. m a c r o ­ carp on New Jersey0 31 0 V. aneustifolium Quebec® 20 100.0 V. anKustifolium Michigan 58 79.2 vaccinii V. corymbosum Nova Scotiad 20 100.0 vaccinii V. corymbosum Michigan 183 82.3 Vaccinii vaccinii vaccinii . Isolate Isolate 0 Isolate Isolate supplied supplied supplied supplied by by by by E. R. A. C. Smerlis. J. Friend. Stretch. L. Lockhart. 6 0 *14. 117 counted whereas only 2 new lesions were observed during the 3pring of 1968. The field was flooded by an adjacent lake during April-July, troyed. 1967 and aH apothecia were d e s ­ Destruction of apothecia probably explains the low level of infection in 1968. Definite infection periods were determined during 1968 (Pig. 26). The Infection Index in Field B was greatest for plants exposed during May 29-July 10 and plants exposed during August 21-0ctober 9* Infection Indices in Field A were similar except that plants exposed during July 10August 21 also had a high Infection Index. precipitation records Examination of ($9) from nearby weather stations showed that it rained for several days before and after August 21 in Field A, but not in Field B (Fig. 27). No sections taken from control plants were infected with Godronia. Conidia were most abundant during April-June in both fields. Spores were observed washing down stems during rains in late May. July. Ascospores were not mature until mid- Apothecia were normally closed (Fig. 16-A) until they were placed in moisture chambers for several hours (Fig. 1 6 - C ) . Although conditions influencing ascospore discharge by Godronia have not been studied, it was recently shown that l\. hours of rainfall were necessary before ascospores of Scleroderris lagerbergii Gremmen (£5. lagerbergerii has 118 leathery apothecia closely resembling those of G o d r o n i a ) were discharged (1^-9) • Spore discharge was more related to rainfall than to relative humidity or temperature (5>0). It seems likely that ascospores of Godronia are also dis ­ charged during periods of rain. Assuming, then, that asco­ spores and conidia are dispersed during rainy periods, several conclusions regarding infection periods can be made. The h i g h Infection Indices on plants exposed in Field A during exposure periods 3 and lj. (Fig. 26) probably resulted because both sets of plants were exposed during the same major infection period. Ascospore infection of plants in Field A probably occurred between August 12 and September 12 since this was the only period of rainfall between 10 July and 9 October. In Field B, infection via ascospores probably occurred between August 30 and September 10 . Infection by conidia probably occurred during the spring and early summer. In Field A, infection by conidia was probably concentrated during rains between May 13 and July 2. Precipitation was not as frequent in Field B and conidia dispersal must have occurred between May 29 and July 2. It seems likely, therefore, that spring and early summer infection by conidia occurs between April and July with heaviest infection occurring in June. Ascospore infec­ tion of plants occurs between mid-August and mid-September. 119 Field A Field B No. sections infected 120 2 3 4 Exposure periods Pig. 26. 5 Number of sections infected with Godronia (i.e., number sections yielding G o d r o n i a ) when 500 sections from each group of exposed plants were placed on h PDA. Exposure periods were: 1 = 23 April-29 May; 2 = 29 May-10 July; 3 = 10 July21 August; lj. = 21 August-9 October; 5 = 9 October3 December. Field A was located in upper Michigan and Field B in northern lower Michigan. 120 Infection Index* 61 Infection Index * 6 0 .EUO- O ct9 Day Infection Indexe 6 (Infection Index* 24 Day of m ontl Pig. 27. L Oct8 Amounts of precipitation recorded at weather stations near Field A (upper) and Field B (lower) between 1 July and 9 October, 1 9 6 8 . Infection Index represents the number of sections yielding Godronia when 500 sections taken from each group of exposed plants were placed on Hs PDA. Vertical stippled lines indicate dates when plants were placed into and removed from the fields. 121 The conclusions stated above- are consistent with existing data, however studies of conditions favoring spore discharge and infection would add to the value of this hypothesis. Infection sites; Reports of others (16, 37* 73) and personal observations indicated leaf scars were i m p o r ­ tant sites of infection by G . cassandrae f • v a c c i n i i . To determine relative numbers of nodal and internodal infec­ tion sites, random samples of 1- and 2-year-old stems were collected in April, 1968. The numbers of lesions occurring at nodes and internodes were recorded. Isolations were made from lesions to verify presence of G o d r o n i a . As shown in Table 25, l± 3,7 % lesions observed were at leaf scars and 21.7$ leaf scars were infected. Cankers at nodes were generally larger than internodal cankers. Only 28.1$ lesions less than 0.5 cm long were at nodes (Table 27) whereas nodally (Table 27). cankers exceeding 1.5 cm occurred Only 11.2$ internodal infections were observed in April as developed cankers (Table 26) whereas 5U-•1% leaf scar infections Cohoon and Daines Daines (Table 26) were developed cankers. (10), Daines, e t . al. (17)* and* (18) showed that leaf scar infection sites of Fusiooc- cum canker of peach (F. a m y g d a l i . the causal organism of Fusicoccum canker of peach,is not related to G o d r o n i a ) decreased as leaf scars healed. Several experiments were performed to determine when and h o w Godronia infects leaf scars on blueberries. 122 Table 25. Godronia infection 3ites on 1- and 2-■year-old stems collected in April 1968. Number stems collected Field Upper Peninsula Total number lesions % lesions at leaf scars % leaf scars infected 1,689 1*0.1 29-8 Ludington 38 2$6 67.9 27.9 Totals 93 1» 91*5 1*3.7 21.7 Table 26. Percent total infections , nodal infections, and internodal infections by Godronia occurring as lesions of various sizes • Lesion size < 0 . 5 cm 0.5-1.5 cm > 1 . 5 cm % total infection sites .70.5 20.1 9.1* %' total leaf scar infection sites 1*5.9 33-2 20.9 % total internodal infection sites 88.8 10.2 1.0 Table 27. Percent Godronia lesions of various sizes occurring at nodes and internodes. < 0 . 5 cm Lesion size 0.5-1.5 cm > 1 . 5 cm % lesions occurring at nodes 28.1 71.1 91*.1* % lesions occurring at internodes 71.9 28.9 5.6 123 Random samples of stems formed during 1968 were c o l ­ lected on October 10, 1968. Petioles of leaves, buds, necrotic leaf tissues, and stem internodes were placed on H PDA to isolate G o d r o n i a . buds, necrotic leaf tissues, invaded by G o d r o n i a . As shown in Table 28, petioles, and internodal tissues had been Attempts to isolate Godronia from leaves and petioles in August had failed Table 28. lateral (Table 28}. Infection of necrotic leaves, petioles, buds, stem internodes by Godronia. Petioles Date Buds Leaves and Internodes 30 Aufsust No. sampled % infected 30 0 30 0 — - — 10 October 136 k k 'Q No* sampled % infected 138 76.8 26 80.7 153 67.9 Petioles f r o m which Godronia was isolated were slightly reddened, but otherwise normal in appearance. showed no external signs of infection. had been initiated from wounds. Buds Necrosis of leaves Godronia was isolated from stem internodes before lesions had developed. The only sign of infections were small zones of gray colored tissue. Observations of similar stems were made in the field D e c e m ­ ber 3, 1968. At this time minute red lesions were observed 12k (Pig. 1 - A ) . Godronia was isolated from most, but not all such lesions. Several stems from the October sample were wrapped in moist paper towels, sealed with aluminum foil, and placed in a 2 C cold room. Well developed lesions w i t h pycnidia occurring nodally and internodally were observed when stems were examined after 6 months in the cold room (Pig. 1 3 - A ) . It seemed that nodes were infected via petioles or buds. Infection of leaf scars via petioles was studied. Infection of leaf scars prior to leaf drop; Three L|.-year-old plants grown in the cold frame were sprayed in October 2-3 weeks before leaf fall with a suspension of G o d ­ ronia conidia in water. The plants were covered with plastic bags and incubated for 3 weeks in the 16 C mist chamber. Plants were returned to the cold frame for the winter. Num­ bers of lesions occurring at nodes and internodes were recor­ ded in April. A control plant was sprayed with water. The number of lesions on each plant varied (Table 29). However, 8 3 .3 $ of all lesions developed around leaf scars. Infection of healing leaf scars; The ability of Godronia to infect healing leaf scars was studied in the following manner. Leaf blades were removed from petioles on actively growing plants. Petioles were inoculated with suspensions of Godronia conidia in water or mycelium in blocks of h PDA at 0, 1, 2, and $ days after removing leaf 125 blades. Inoculation sites were wrapped and plants were placed in the 18 C mist chamber for 10 days. Following incubation plants were grown in an 18 C greenhouse. Con ­ trols were plants inoculated with water or blocks of Table 29. PDA. Infection of leaf scars by Godronia when suspen­ sions of conidia in water were sprayed on plants 2-3 weeks before leaf drop. Plant number Total number lesions Number lesions at leaf scars 1 17 12 2 10 7 3 39 36 0 — Lt-{HpO con­ trol ) Totals 66 55 = 83-3# As shown in Table 30* the percentage of leaf scar infections decreased as the length of time after removing leaf blades increased. Table 30. Infection of leaf scars via petioles by Godronia at various times after removing leaf blades. D a y s after removing leaf blade Number petioles inoculated % leaf scars infected 0 kl 3U--0 1 5U- 31.lt 2 38 7.8 5 72 5.5 126 Infection of healed leaf a c a r s ; Healed leaf scars on dormant 3-year-old plants in a cold frame were inocula­ ted in February 1968, using Godronia mycelium in blocks of PDA as inoculum. Following inoculation plants were incu­ bated for 12 day3 in a 18 C mist chamber and then returned to the cold frame. Leaf scars pierced a single time with a sterile needle and inoculated with G o d r o n i a ; and nonwounded leaf scars inoculated with blocks of PDA served as controls. Lesions developed from wounded leaf scars inocula­ ted with Godronia within a few days following inoculation. No lesions were discernable at healed leaf scars for 1 year. However, between February 25 and May 3 t 1989* lesions developed at 3 0 .2 # of 129 inoculated nonwounded nodes. Since inoculum had been In contact with both leaf scars and axillary buds, it was not determined which served as the infection court. PDA were lesioned. None of the nodes inoculated with hk This is the first report of latent infection by G o d r o n i a . Infection of internodes: Two experiments were designed to demonstrate infection of nonwounded stem inter­ nodes by G o d r o n i a . Plants actively growing in the greenhouse were sprayed with suspensions of conidia in water, with plastic bags, chamber. covered and incubated for 2 weeks in a 18 C mist No infections developed on these plants when they were grown in the 18 C greenhouse. 127 Nonwounded stem internodes on two ij.-year-old dor­ mant plants were inoculated using the procedures outlined in previous experiments. Five of 90 inoculated internodes were infected by G o d r o n i a . Lesions did not develop on n o n ­ wounded 3tem internodes inoculated with blocks of \ PDA. All internodes which were wounded and inoculated as con­ trols were infected. * It was concluded from these experiments that leaf scars are probably infected via attached petioles or axil­ lary buds. This conclusion is consistent with data obtained when plants were exposed to natural inoculum. That infec­ tion takes place between mid-August and mid-September and that leaf scars are invaded via attached petioles indicate that fingicides applied by other workers (16, 35> 73) were uneffective, at least in part, because they were applied long after infection had occurred. P A R T XII. PATHOLOGICAL HISTOLOGY. Methods and Materials Symptomatology of cankers caused by Godronia showed that lesions on 1- and 2-year-old steins were first visible as minute red spots on internodes and as water soaked or necrotic leaf scars. Necrosis was observed to begin in the centers of incipient internodal lesions. Bands of reddened tissue were often observed along the edges of necrotic tissues. Development of lesions caused by Godronia was stu­ died histologically. Lesions or cankers of various sizes on 1- to 3-year-old stems were collected. Diseased tissues were divided into categories based on the age of the lesion: a) type 1 - incipient lesions less than 2.0 m m in diameter (Pig. 1-A, B); Type 2 - 0.£-l.0 cm long, elliptically shaped lesions devoid of pycnidia (Pig. 1-B, C); Type 3 -]>1.0 cm long, elliptically shaped lesions covered with pycnidia (Fig. 1-D; 2-A, B ) ; Type 1*. - discolored xylem from stems beginning to wilt. Callus tissue associated with cankers was also studied histologically, but no attempt was made to follow stages of callus development. All tissues were neither fixed nor stained. gential and transverse 10-l£ 128 Tan­ thick sections were cut with 129 an experimental microtome (29). Living sections were mounted either in water or glycerin diluted to with water (v/v). Twenty to 30 sections were cut through both the centers and edges of lesions. At least £ lesions of each Type were examined on each of 2 sampling dates. collected only during June. Callus tissue was Healthy tissue from uninfected branches of the same ages as cankered branches served as controls. Samples of lesions were placed on hs PDA and God- ronia was grown from stems with each of the symptom cate­ gories . Observations were made with a Wild-Heebrugg M-20 compound light microscope equipped with a K o d a k II 3$ m m camera. Photomicrographs were taken on highspeed 3$ nun Kodak Ectachrome X film. 130 Pig. 28. Transverse sections of h e a l t h y blueb e r r y stem and stems infected w i t h Q o d r o n i a . All sections cut with an experimental microtome (29) and mounted in water. A) Section of noninfected 1-year-old stem (2$0 X). (a) cuticle, (b) stoma, (c) substomatal c h a m ­ ber, (d) tightly packed cortex parenchyma, (e) air channels, (f) loosely pa c k e d cortex p a r e n ­ chyma, (g) pericyclic fibers, (h) phloem, (i) cambium, (j) xylem. B) Incipient necrosis (b) below stoma of Type 1 lesion (200 X). C) H y p h a in air channel of cortex D) Hy p h a e in air channel of cortex near reddened cortex parenchyma (ij.00 X). E) H y p h a in air channel near reddened cortex parenc h y m a (800 X). (a) in center (000 X). 131 132 Pig, 29. Sections of stems infected w i t h Godronla (A-P mounted in water; G and H mounted in 5 0% g l y c e r i n ) . Radial section showing hyphae in air channel of cortex (800 X). B Hyphae growing along surface of living cortex parenchyma cells (800 X ) . Transverse section through Type 3 lesion show­ ing hy p h a e in dead cortex parenchyma (800 X). Transverse section through Type 3 lesion show­ ing deposits in cortex parenchyma (200 X). E Section through center of Type 2 lesion showing general mecrosis and hyphae (arrow) in air channel (200 X), Longitudinal section through pycnidium of G o d ­ ronia in necrotic cortex tissues (200 X). Radial section showing hyphae in vessel of discolored xylem of wilted stem (800 X ) . H Transverse section through discolored x y l e m of wilted stem showing brown deposits (a) and hy p h a (b) in vessels (800 X). 133 J 131* Pig. 30. Sections of diseased blueberry stems. (A, B* C, and P mounted in water; D, E, and G mounted in 5 0 # glycerin. A) Hyphae in necrot±tr tissue of callus {000 X). B) Hyphae and necrosis in bud tissues C) Disarranged vascular tissue in 8 -D callus (200 X ) . D) Brown deposits between 2 vessels in d i s ­ colored xylem of wilted stem (800 X). E) Hypha growing through scaliform perforation plate of vessel in discolored xylem of wilted stem (800 X ) . P) Brown deposits in discolored xylem of wilted stem (200 X). G) Possible tyloses in vessel of discolored xylem of wilted stem (800 X). (200 X). Experimental Results The only detailed study of blueberry stem anatomy was published by Wasscher (6 3 ). The following observa­ tions (Pig. 28-A) of transverse sections of 1-year-old healthy 3tems of an unnamed blueberry variety agreed with Wasscher's (6 3 ) data. The epidermis was protected by a 10-l£ ju thick cuticle. The cuticle was interrupted by numerous stomata which were bordered by 2 thin-walled guard cells. The outer cortex consisted of a fj-12 cell layer of tightly packed parenchyma cells which were filled with chloroplasts. In transverse sections the remaining cortex was spongy in appearance due to presence of longitudinal air channels, which according to Eck (2Ij.), "are so oriented that 2 ducts are adjacent to one another along the radial axis." The cortex and vascular tissues were separated by a layer of thick-walled fibers called "pericyclic" fibers by Wasscher (63). Observations of transverse and tangential sections cut through the center of Type 1 lesions showed that epider­ mal cells were unaffected and that parenchyma cells of the cortex immediately below stomates were the first to die (Pig. 28-B). Moribund cells were filled with dark brown deposits and hyphae could not be seen in the cells. were, however, Hyphae observed among cortex parenchyma adjacent to 136 137 pockets of necrosis (Pig. 28-C, D, E ) . The chloroplasts of parenchyma adjacent to necrotic cells turned red. Cells with red chloroplasts were often observed 16-20 m in ad­ vance of growing hyphae. Hyphae were most abundant within dead cells and in longitudinal air spaces (Pig. 28-C, D, Ej 29-A, B, C ) . channels. Hyphae seemed to advance primarily via air The fungus often grew along the surfaces of living cortex parenchyma cell 3 (Pig. 29-B), but penetra­ tion of living cells was not observed. Hyphae were usually found in dead cells of the cortex (Pig. 29-C), but not inside of living cells. Air channels and dead cells of the cortex were filled with brown deposits and the cell walls turned brown (Pig. 29-D). All cortex tissue beneath Type 3 lesions was necro­ tic and permeated with hyphae (Pig. 2 9 - E ) . Pycnidia were formed in outer layers of necrotic cortex tissue (Pig. 2 9 - F ) * The xylem of stems with Type 3 lesions was starting to turn brown. Hyphae, possible tyloses, and brown deposits were observed in vessels of the xylem (Pig. 29-G, H ) , but were confined to outermost cells. Hyphae, tyloses, and deposits were common in the vessels of wilted stems (Pig. 29-H, 30-D, E, P, G). Thirty to lj.0# of all vessels in a given 1 0- 15 u thick transverse section were occluded (Pig. 30**P)» Observations of tangen­ tial sections showed that cell walls of vessels, ray paren­ chyma and fibers turned brown. Hyphae grew longitudinally 138 through vessels and from cell to cell by growing through scaliform end plates oi‘ vessels (Pig. 30-E). vessels were often clogged with brown deposits The ends of (Pig. 30-D). Buds associated with lesion Types 2 and 3 were necrotic and thoroughly permeated with hyphae (Pig. 30-A, B). Callus tissue was composed to disarranged parenchyma and vascular tissue (Pig. 30-c). luses along the edges of cankers Hyphae were common in cal­ (Pig. 3 0 - A ) . DISCUSSION Koch's Postulates were fulfilled with P_. vaccinll and G. cassandrae showing that both fungi cause cankers and stem blights of blueberries in Michigan. The diseases will be called Phomopsis canker and stem blight and Godronia (Fusicoccum) canker and stem blight, respectively. Phomopsis canker and stem blight is used in lieu of Phomopsis twig and cane blight because the former name is more descriptive of the disease as it occurs in Michigan. Godronia (Fusicoccum) canker and stem blight is preferred to Fusicoccum canker for several reasons. Fusicoccum is an invalid name for asexual states of Godronia (28). Use of Fusicoccum canker might imply that a taxonomic relation­ ship exists among asexual states of Godronia and the causal organisms of diseases caused by Fusicoccum s p p . relationship does not always exist. Such a For example, Fusicoc- cum canker of peach is similar in appearance to Godronia (Fusicoccum) canker and stem blight of blueberry, but is caused by a fungus unrelated to Godronia (10, 17, 18). Apothecia of Godronia are apparently important sources of inoculum, and are easily found in severely affected fields. Therefore using the name of the sexual state of the organism appears justified. Since few publications (3* 16 > 30, 39* 139 iij.o 61, 70, 73) have used the name Fusicoccum canker, it will be less confusing in the final analysis, descriptive Godronia to use the more (F u s i c o c c u m ) canker and stem blight of blueberry. Symptoms associated with Phomopsis and Godronia on 1- and 2-year-old stems in Michigan were in most respects identical to those reported by others (3 # 1 6 , 3 5 # 3 7 # 3 9 , 61, 6 6 , 67# 70, 73). One significant difference was that in Michigan both fungi caused lesions and stem blight symptoms on 1- and 2-year-old stems. Also many lesions caused by Phomopsis expanded and caused blight symptoms on artifically inoculated stems. direct contrast to earlier observations This is in (6 6 ) that only localized lesions followed inoculation of woody stems. In Michigan, local lesions caused by Godronia and stem blight symptoms caused by Phomopsis, respectively, are usually diagnostic characters of the 2 dieseases on 1 - and 2 -year-old stems. Diagnosis in the field is more difficult on older stems. Diagnostic lesions and discoloration caused by G o d ­ ronia do not occur once bark is formed. Developed cankers and stem blight are the only visible symptoms on older stems. Cankers caused by Godronia tended to be short and wide, whereas Phomopsis cankers were usually long and narrow. This distinction does not always hold, however, and isola­ tions from infected stems are often necessary to confirm diagnoses made in the field. Blight symptoms caused by 1U-1 Godronia and Phomopsis are easily differentiated if pycnidia are present on affected stems. However, isolations are necessary for positive diagnosis when fruiting bodies do not occur. The association of Godr o n i a . and to a lesser degree Phomopsis. with certain kinds of calluses and the results of girdling experiments suggest that calluses at the edges of cankers and wound type calluses probably result from the alow girdling action of cankers caused by these fungi. Cal­ luses along the edges of cankers located in the crown of affected plants and calluses along cankers at the bases of lateral branches may be similarly caused. The cause of cal- lusing along the length of affected stems remains to be determined. It is important to stress that Phomopsis was not associated with calluses similar to those described by Brown (7). Additional studies of the callus syndrome are necessary to identify positively the causal agents. Wilted stems (flags) were the most conspicuous symptom of both diseases. Histology of discolored xylem of flagged stems infected with Godronia indicated that stems wilt as a result of vascular occlusion rather than due to girdling. In other words, the xylem is invaded by the gus before stems are blighted. In this respect, fun­ the 2 diseases are probably similar. Based on observations that Godronia (Fusicoccum) canker and stem blight is generally more severe and more llj.2 common in areas where cool climates prevail it has been sug­ gested that plants are predisposed to the disease by cold injury (lj.0, $ h ) • Effects of cold injury on Godronia (Fusi­ coccum) canker and stem blight were not studied. However, increased severity of the disease at 2-8 C when inoculated plants were grown at various temperatures, Indicates that low temperatures and dormancy predispose blueberry plants to this disease. These data suggest that the disease is more severe in cool climates because there are longer periods during the year when growth of the fungus is favored over growth of the host. Whether the fungus invades the xylem directly when plants are dormant or whether it must first grow to vascular tissues leading to axillary buds and leaf scars remains to be proven. Knowledge of how Godronia invades the xylem of affected plants could have practical implications. example, For in upper Michigan, Godronia causes cankers on Rancocas variety as often as on other varieties. Cankered stems of Rancocas, however, do not wilt as often as those of other varieties. In one upper Michigan, field, for example, wilted stems were found on 6 .6 # of 300 Rancocas plants observed whereas 3 2 .2 # of 1 ,014.7 plants of other varieties were flagged. This observation suggests that Rancocas is infected as often as other varieties, but G o d ­ ronia does not as readily invade the xylem of Rancocas plants. The reason for this phenomenon is not know. One H43 possibility, however, is that Godronia can not penetrate the protective cylinder of pericyclic fibers around the vascular cylinder and invades the xylem only via vascular elements leading to leaf scars and axillary buds. Available evidence suggest that Godronia is indeed a weak parasite and can invade xylem tissues only when the plant is relatively inactive metabolically. Increased severity of the disease on plants grown under conditions favoring dormancy, for example, supports this conclusion. In addition, histological observations indicated that G o d ­ ronia can not penetrate living cells and that growth of the fungus in the living cortex progresses primarily through the longitudinal air channels. Internodal lesions observed in April walled off during the summer as did lesions result­ ing from artificial inoculations. These observations indi­ cate that infections occurring during the early summer and infections occurring along stem internodes may not result in development of blight symptoms. Existence of a thick layer of pericyclic fibers, fewer air channels, or few leaf scar infection sites could account for the tolerance of Rancocas. These possibilities and the way in which Godronia invades the xylem should be investigated. Distribution of Phomopsis and Godronia in Michigan and Indiana may be related to temperature. Godronia, for example, grows poorly at temperatures above 25 C and was not found in southern areas where warmer temperatures Di)i T I prevail. Phomopaia which grows well at 2f?-30 C and caused cankers on plants artificially inoculated when temperatures exceeded 30 C, was common in warmer areas. That temperature plays a role in distribution of the diseases is supported by studies of a dieback disease of cranberry which suggested that warm temperatures favored growth of Phomopsis over that of Godronia in diseased cranberry stems (26). Studies of the effect of temperature on disease development, and the pr o ­ duction, dispersal, and germination of spores of these fungi may show that temperature indeed limits the distribution and severity of these diseases in Michigan and Indiana. Isolation of Godronia from plants exposed to natural inoculum at various times during the 1968 season provided the first experimental evidence for the occurrence of 2 infection periods during the growing season. Infection during June was probably due to conidia since conidia were observed washing down stems during this time and ascospores were immature. Disappearance of ascospores from apothecia corresponded to the mid-August to mid-September infection period. The probable disease cycle of Godronia (Fusicoc­ cum) canker and stem blight is outlined in Fig. 31* Data indicating that there are 2 infection periods suggest that attempts to control Godronia (Fusicoccum) can­ ker and stem blight have failed, at least in part, because compounds were applied at the wrong time (i.e., either before or after actual infection periods). In Nova Scotia (3$), 11*5 Pig. 31. Diagram of canker and defined as during the the probable disease cycle of Godronia stem blight. Primary infection is the first infection of new plants growing season. U|7 for example, fungicides were applied during the last week in September, mid-October, and in early May. Assuming that the disease cycle is similar in Nova Scotia, each of these applications could have missed actual infection periods. It is interesting to note that although most late infection probably occurred before September 1$, 1968, lesions were not visible until December. Lesions did not develop from axillary buds and healed leaf scars until a year after inoculations. Appearance of lesions, therefore, can not be used as an accurate indication of when infection occurs. Circumstantial evidence strongly suggests that Godronia infects leaf scars via attached petioles, but direct proof is lacking. The fungus was isolated from sampled petioles following the late summer infection period which occurred I4. to 8 weeks before leaf drop. Little infection occurred on plants exposed to natural inoculum (i.e., 9 October - 3 December) during and after leaf drop. More leaf scars were infected when attached petioles were inocu­ lated at 0 and 1 day after removing leaf blades than when petioles were inoculated at 2 and $ days after leaf blades were removed. Healed leaf scars were not infected until a year after inoculation whereas wounded leaf scars were infected immediately following inoculation. Actual ingres- sion of Godronia into leaf scars from artificially inocula­ ted leaf blades was not studied. That this kind of experiment 114.8 should be executed before concluding that leaf scars are invaded via attached petioles is suggested by studies of FusicoccuCT canker of peach. These studies showed that F. amygdali infected healing leaf scars (1 0 , 1 7 ), but did not infect leaf scars when leaf blades were inoculated (1 0 ). Evidence for infection of stem internodes via stomates is also circumstantial. Histology showed that incipient necrosis always occurred below stomates. Some infections resulted from artificial inoculations made on nonwounded stem internodes. That $6% of all lesions on 1- and 2 -year-old stems occurred on stem internodes and that infected stems were not wounded suggests that Godronia either penetrates stems directly or invades stomates. Histological observations suggested that Godronia is unable to penetrate living cells. Since the stem is protected by a 10-15 & thick cuticle interrupted only by stomates, in­ fection of internodes of 1 - and 2 -year-old stems via stomates seems likely. All major varieties of blueberries grown in Michi­ gan are very susceptible to Godronia (Fusicoccum) canker and stem blight, whereas only Earliblue variety is severly affected by Phomopsis. Godronia (Fusicoccum) canker and stem blight is, therefore, considered to be the more impor­ tant disease in Michigan. Phomopsis canker and stem blight is, however, a potentially devastating disease. ple, in one well tended field in Indiana, For exam­ over 200 twelve- 349 year-old Earliblue variety plants were unproductive because of the disease. Losses in this field were estimated to exceed $2,000 annually. Godronia fFusicoccum) canker and stem blight is equally as devastating in northern lower and upper Michigan where all plants were infected in several fields. Pathogenicity of isolates of G. cassandrae from Spiraea spp. and V^. angustifolium on V,* corymbosum suggests that infected Spiraea s p p . and V. angustifolium serve as inoculum reservoirs for Godronia canker and stem blight. These hosts are very common in Michigan blueberry growing areas and inoculum from these plants could be an important source of infection in Michigan blueberry fields. Godronia cassandrae was divided into 6 forms accord­ ing to the genera of plants inhabited by each fungus Smerlis (28). (£1) used differences in ranges of pathogenicity to differentiate among forms cassandrae, beticola, and vaccinii. Forms cassandrae and vaccinii were separated by differences in cultural characteristics (5 1 )• In my studies, 2 separate isolates of G. cassandrae from V. macrocarpon did not cause cankers on inoculated blueberry plants confirming earlier observations inoculations made with similar isolates. (3 7 ) of One of 2 isolates from £. calyoulata was pathogenic on blueberry, but only 1 lesion resulted from inoculations with the pathogenic Quebec isolate. Two of I4. isolates from Spiraea s p p . were pathogenic i$o on blueberry. All isolates obtained from V. corymbosum and V. angustifolium were pathogenic on inoculated blueberry plants. These data indicate that earlier interpretations of form vaccinii based on pathogenicity (28, 51) must be investigated further.' More inoculations with single spore isolates of each form must be executed before pathogenicity can be used to differentiate among forms spiraeicola, cas­ sandrae . and vacc i n i i . Variability noted in spore morphology and cultural characteristics of G. cassandrae, especially isolates from highbush and lowbush blueberries, indicates that a large number of single spore isolates must be grown and compared under precisely controlled conditions before these charac­ ters are useful as taxonomic parameters. For example, not all isolates of G. cassandrae from V. angustifolium produced the slime and black mycelium used by Smerlis ate f . vaccinii from f. cassandrae. (51) to separ­ Some isolates, in fact, produced slime only when grown at temperatures exceeding 20 G. Available evidence indicates that there are probably several distinct strains of G. cassandrae with different ranges of pathogenicity. However, before conclusions con­ cerning the taxonomy of strains can be made, pathogenicity and cultural characteristics of more single spore isolates from the various hosts must be studied. In brief summary, 2 canker and stem blight diseases of blueberry are common in Michigan. Godronia (Fusicoccum) 151 canker and stem blight is considered to be more important than Phomopsis canker and stem blight* Godronia cassandrae from Spiraea s p p . and V. angustlfolium were pathogenic on V. corymbosum* Blueberry plants were infected by G. cas- sandrae during June and between mid-August and mid-September. Stomates and leaf scars probably function as infection sites. Circumstantial evidence suggests that leaf scars are invaded via attached peti o l e s . Dormancy and low tem ­ peratures favored development of blight symptoms on arti ­ ficially inoculated blueberries. Histology indicated that stems wilt when the xylem is invaded by the fungus and ves­ sels are occluded by hyphae, various deposits and possibly tyloses. Some calluses on affected blueberries were a s s o ­ ciated with cankers caused by Godronia and Phomopsis. LITERATURE CITED 1. Alexanderf J. V., R. J. Cook and J. A. Bourret. 1965* Fusarlum species associated with cacao galls. Phytopathology 55: 125-126 (Abstr.). 2 . Alexopoulos, C. J. and E. S. Beneke. 1961+. Laboratory Manual for Introductory Mycology. Second printing. Burgess Publishing Co.f Minneapolis, Minnesota. 199 p. 3- Barnes, E. H. and H. C. Tweedie. 1961+. Fusicoccum canker of highbush blueberry in Michigan. Plant D i s . Reptr. 1+8: 6 8 7 - 6 8 9 * 1+- Boyce, John, Shaw. 1 9 6 1 . Forest Pathology. 3rd ed. McGraw-Hill Book Co., Inc., New York. 572 p. 5- Brown, Nellie A. 1931+- Phomopsis gall on V i b u r n u m . Phytopathology 21+: 1119-1120. 6. Brown, Nellie A. 1937* The tumor disease of oak and hickory. 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