z .qu Liv 1... . . . .74. nun—n. I ‘ ”9.5V?! . 2r (anti... 7 . .r :21. a. y i!!!«...wm. .1: , I!" .23... . o.!.».r.ld S. An : N ESTIG NV. fl .... ., an} ifs...“,.,...§~...J_..,.$ ix. £59.. w. .. $5.555? a3. ..;5u.§. l‘ LIBRfi R 3’ “ Mulligan State University [HESIS This is to certifg that the thesis entitled INVESTIGATIONS OF INTERNAL BARK NECROSIS IN DELICIOUS APPLE TREES presented by ‘ Timothy Eugene Crocker has been accepted towards fulfillment of the requirements for th D. degree in HortiCUlture 1 % flew/52¢ Major professor ' . d 7fl Date €25 ’1 / /€ 0469 ABSTRACT INVESTIGATIONS OF INTERNAL BARK NECROSIS IN DELICIOUS APPLE TREES By Timothy Eugene Crocker Internal bark necrosis (IBN) often referred to as l'measles” was first described in detail by Hewitt and Truax in 1912. This disorder occurs predominantly on trees of the Delicious cultivar. The disease may result in a considerable reduction in growth and/or death of newly planted trees. Many causative agents have been suggested as the cause of IBN: (l) a toxicity of the elements Mn, Fe, Cu, Co, Al, Zn, or Ni, (2) a toxic combination of two or more of these metals or (3) a deficiency of B. An experiment was designed in 1967 to induce IBN on Delicious trees. The trees were grown in sand culture with two levels (normal and low) of Ca in combination with two levels (100 and 200 ppm) of Mn, Fe, Cu, Al, a mix of 50 ppm of each metal, and a minus B treatment. Only trees receiving the Mn or minus B treatments developed ibN. Trees treated with other treatments had a greater than Timothy Eugene Crocker normal leaf accumulation of each respective metal, but there were no symptoms of IBN. ' A I An experiment was conducted in 1968 to determine the concentration of Mn associated with a severity rat— ing of IBN. Trees were grown in sand culture with nor— mal and low levels of Ca in combination with five levels (0, 25, 50 75, and 100 ppm) of Mn. The severity of IBN increased with increasing leaf Mn content. A leaf Mn value of 500 ppm was established from results of this experiment as the value of Mn above which IBN may become severe. An experiment was conducted in 1969 to determine if the severity of IBN occurred to a greater degree on spur—type than on Standard Delicious trees. Trees of both growth characteristics were grown in sand culture and received treatments of 50,100, and 150 ppm Mn. Nei— ther severity of IBN nor leaf Mn were found to differ significantly for tree type. Terminal growth was signi— ficantly greater for the standard type trees. The use of a corrective application of lime for TBN was studied in 1968. Five concentrations 0, 5, 10, 30, and 50 pounds of dolomitic hydrated lime per 100 gal— lons of water at three rates, 1, 2 and 3 gallons were applied at the base of the trees. Leaf Mg, soil Mg, soil Ca and soil pH were significantly increased for treatments. No significant differences in IBM or available Timothy Eugene Crocker soil Mn occurred-with lime treatments. However, IBN was not severe in any 'of the planlings studied. ' applied at .5 and 1 1b.. NHuNO (NHM)2SOA and.NaNO , 3 ’ 3 -actual N per tree were compared to no N on the occurrence of IBN on Delicious trees. NHuNO _and (NHu)2soLl in—' 3 ‘creased the soil acidity and seil Mn significantly from the control (no N). No significant differences were" observed for leaf N or IBN rating. The microprobe X—ray analyzer was used to analyze thin sections of bark tissue affected with lBN. Mn and Ca were found in larger quantities in the necrotic areas than non—necrotic areas of tissue from trees treated with excess Mn. Minus B induced IBN lesions had a larger Ca concentration. K and P were found in smaller quanti- ties within the necrotic lesions than non—necrotic areas for both Mn and minus B induced IBN. ObServations and analysis of tissue representing symptoms called IBN in areas outside of Michigan, indica— ted that the symptoms were unlike those induced in this study and, apparently were related to some factor not identified rather than excess Mn or deficiency B. INVESTIGATIONS OF INTERNAL BARK NECROSIS IN DELICIOUS APPLE TREES By Timothy Eugene Crocker A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1970 ACKNOWLEDGMENTS The author expresses his sincere thanks and appree ciation to Dr. A. L. Kenworthy for his assistance and guidance in Carrying out the experimental work and pre— paring the manuscript; to Dr. H. P. Rusmussen for his assistance and advice in the electron micrOprobe analyses and preparation of the manuscript; to Drs. C. M. Harrison, Jerome Hull Jr. and H. M. Sell for their suggestions in editing the manuscript. The financial support of the NDEA Title IV fellow— ship is gratefully acknowledged. TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION . '. REVIEW OF LITERATURE . . . . . EXPERIMENTS Experiment I . . . . Results . . . . . . . . Discussion Experiment II. Results Discussion Experiment III Results Discussion Experiment IV. Results Discussion Experiment V Results Discussion Experiment VI. Results Discussion SUMMARY BIBLIOGRAPHY. Page ii 'iv Vi ll 19 22 2A 31 32 3M 35 an 147 53 59 63 6A 67 LIST OF TABLES Table Page 1., Mn content of leaf and bark tissues as affected by varying concentrations of Mn and Ca . . . . . . . . l3 2. B content of leaf and bark tissues as affected by varying concentrations of Mn and Ca . . . . . . . . . l3 3. Fe content of leaf and bark tissues as affected by varying concentrations of Mn and Ca . . . . . . . . . . . l6 4. Ca content of leaf and bark tissues as affected by varying concentrations of Mn and Ca . . . . . . . . . . . l8 5. Al content of leaf and bark tissues as affected by varying concentrations of Mn and Ca . . . . . . . . . . . 20 6. IBN rating scheme for determining the severity of IBN on apple trees . . . . 23 7. Average IBN rating on Delicious trees grown with varying levels of Mn and Ca for two seasons . . . . . . . . . 2A 8. Rating of IBN on Delicious trees receiving varying levels of Ca and Mn after one growing season . . . . . . . . . 25 9. Leaf concentration of Ca, Mn and rating of IBN as affected by normal and low Ca levels . . . . . . . . . . . 26 10. Standard versus spur—type Delicious apple trees receiving varying levels of Mn (150, 100 and 50 ppm) on the occurrence of IBN . . . . . . . . . . . . 33 iv Table ll. l2. 13. 14. 15. l6. 17. 18. 19. 20. 21. IBN rating and Mn leaf concentration of standard and spur—type Delicious apple trees as affected by three concentra- tions of Mn (150, 100 and 50 ppm) Mn content of leaves from newly planted young Delicious trees (sampled the year of planting) . . . . . . . Mn leaf content of young Delicious trees in nursery row . . . . . Leaf analysis values and IBN rating of Delicious trees treated with lime slurry solutions. Existing orchards . . Soil analysis results from Delicious trees treated with the lime slurry solutions. Existing orchards . . . . . . Soil analysis results from Delicious trees treated with lime slurry solutions. Existing orchards . . . . Leaf analysis values and IBN rating for planted Delicious trees treated with the lime slurry solutions. Newly planted trees . . . . . . . . . . . Soil analysis results for planted Delicious trees treated with the lime slurry solu— tions. Newly planted trees . . . . . Leaf composition and IBN rating of trees receiving different sources of N. Exis— ting orchards. . . . . . . . . Soil analysis results of trees receiving different sources of N. Existing orchards . . . . . . Leaf nutrient contents of trees with "measle" symptoms from different areas Page 3A 38 39 39 no Al Al u2 “3 AA 63 Figure 1. AA. AB. LIST OF FIGURES Delicious trees in second year of heavy metals and minus B treatments . Leaf Mn content of Delicious trees corre— lated with IBN rating observed after one year of Mn treatment . . Average rating of IBN for low Ca, normal Ca and control observed in October, March and June , , Line profile analysis of B deficient bark demonstrating the distribution of Mn Line profile analysis and X—ray oscillo- gram of normal bark demonstrating the distribution of Mn . . . . . . . . Line profile analysis and X— —ray oscillo— gram of IBN bark demonstrating the dis— tribution of Mn . . . . Bark tissue of apple tree grown with a high Mn treatment and sectioned by using the cryostat procedure. . . . . Bark tissue of apple tree grown in minus B nutrient culture and sectioned by using the cryostat procedure. Symptoms that have been called ”measles" vi Page 1A 27 29 49 A9 51 54 56 61 INTRODUCTION The popularity of the Delicious apple variety and its sports has been increasing during the last decade in all major apple growing areas of the United States and in many foreign countries. In Michigan, Delicious com— prises 30 per cent of the present production, and accounts for 35 per cent of the trees in non—bearing orchards. The production of apples in North Carolina is now 51 per cent Delicious. The same is true for apple production in Washington and other apple—producing areas. Delicious is susceptible to a disorder known as internal bark necrosis (IBN) or "measles”. Hewitt and Truax (22) in 1912 were the first researchers to describe the disorder in detail, and they applied the name ”apple measles". The disorder is worse on Delicious than on other major apple cultivars. Internal bark necrosis may result in the death of newly planted orchards, but more often results in reduced growth and an economical loss due to delayed production because of reduced tree growth. Recent reports on internal bark necrosis have indi— cated various discrepancy opinions as to its cause. Many investigators believe it is associated with a manganese excess or boron deficiency. Still others surmise that it could be excessive amounts of other metals such as Al, Fe, Cu, Co, Zn or Ni. Other researchers suggest an accu— mulation of all the heavy metals, of spray oils_or of the expressed symptoms of a latent virus. V To resolve the confusion, experiments were conduc— ted to determine if it was a heavy metal, a combination of metals, or boron deficiency. Control recommendations were tested to determine the best control practices. Studies were undertaken, also, to determine if there was an accumulation of nutrients in the necrotic areas of the bark tissue. REVIEW OF LITERATURE Internal bark necrosis (IBN) or ”measles” have been reported on Golden Delicious, Grimes, Jonathan, McIntosh, Rome, Northwestern Greening, Stayman, York, and King David (8,30), and from personal observation on R. I. Greening. Nagai, et. al. (26) has reported IBN in Delicious and Roll trees in Japan, and Atkinson and Roberts (3) have reported the symptoms in New Zealand on Delicious. IBN symptoms usually develop a year or two after planting, occurring on the lower branches and trunks of the trees. In very severe cases, the symptoms can appear on current seasons growth, causing severe stunting and‘ eventual death of the tree. Many trees affected with IBN recover and grow normally after a few years. But some trees remain weak and stunted, never resuming nor— mal growth. Berg, et. a1. (8) in West Virginia has observed three types of stem lesions characteristic of IBN——the pimply, the oedematous, and the minute superficial le— sions. The pimply lesions were the most common and found in association with the other two types when they appear. The pimply lesions developed from small necrotic areas which originated deep in the cortex or periderm. Later these areas became encysted within the bark by development of an enclosing layer of suberized cells. The first externally Visible indication of this condi— tion was raised points that appeared on the epidermal surface. These areas increased until the bark took on a characteristic rough surface with numerous cracks and splits. Oedematous lesions occurred most commonly at the base of the trees. The bark of the oedematous lesions became swollen and had a water—soaked appearance. The outer periderm over these swellings split parallel to the axis of the stem and became loose appearing as a series of tan papery sheets covering the unruptured swollen portions. Minute superficial lesions appeared as darkened points on the bark, which were perceptible to the touch, and were confined to the terminal portion of the current season's growth. Berg, et. a1. (7,8) and Shannon (32) reported a yellowing of the leaves sometimes as intervenal chlorosis and sometimes as large or small yellow areas. Affected leaves often abscised prematurely. Many investigators associate an excess of mangan— ese with the disorder (1, 2, 6, 7, 8, 13, 1“, 15, 20, 21, 24, 26, 28, 31, 32, 33, 39) while some do not agree that exCess manganese is the cause or only cause of IBN (10, 20, 39). Nagai, et. el. (26) has reported that the mang— anese content of trees grown in sand culture varied with rootstocks, and that two to three—year wood contained a higher level of manganese than did the current season‘s wood. Berg, et. a1. and Orton, et. a1. (8,28) disclosed that the manganese content of bark was less than found in the leaf; but Nagai, et. a1. (26) found bark higher in manganese content than that found in the leaves. Rogers, et. a1. and Berg, et. a1. (31,8) have shown IBN to be greater when ammonium nitrate or other acidify— ing sources of nitrogen were applied; on the other hand, Cahoon, et. a1. and Hildebrand (10,23) have shown little response to sources of nitrogen. Boron deficiency has similarly been implicated as a cause of IBN (9,24,32, 35, 37, 39), but Berg and Clulo (6) were unable to induce the symptoms with a nutrient solution absent of boron. However, some investigators have shown a response from applied boron (17,24) in cor- recting the disorder. Still other investigators support the evidence that IBN may be a manganese toxicity or a boron deficiency (4, 32, 39). Shannon (32) has induced the symptom with iron. Berg, et. a1. (8) also suggested that iron may be associated with the disorder. Zeider and Kink (39) found that high iron content seemed to delay but not prevent the symptoms caused by manganese excess. He found that 'a 50 ppm iron treatment gave a terminal resetting and stunting of the trees. Forshey (20), in New York, in— duced the symptoms with high levels of manganese and also induced the symptoms by injections of iron, aluminum, copper, zinc, cobalt and nickel. Cobalt was as effective as manganese in inducing the symptoms, but the other ele— ments gave a lesser response. Wave, et. a1. (36), in Washington, stated that copper deficiency symptoms are very similar to those of the early stages of measles. Still other investigators (10, 20) feel that the condi— tion may be aluminum toxicity. Shannon (32), in a histological study, showed that deficiency symptoms of boron and excess symptoms of iron and manganese exhibited the same basic manifestation. His manganese symptoms for IBN agreed with the symptoms for IBN given by Berg, et. a1. (8) and Clulo (4) to be manganese toxicity. Wave and Stiles (36) were able to produce a measles— 1ike symptom using superior oils, but with this condition the pimples or raised areas are associated with the lenti— cels rather than those occurring in smooth areas of the bark as generally observed for IBN. Zwick, et. a1. (40) supported this theory and observed a definite proliferation or corkiness of lenticels in the epidermis of the bark of young pear trees. These conditions have been observed on young Delicious trees by DoWning (l6). Cheney et. a1. (11) reported the symptoms of "pustule—canker", a graft transmissible bark disease of ”Red Delicious" apple, as closely resembling those of measles and blister bark. But he stated that pustule— canker differed from IBN in that the former did show ter— minal die—back. He concluded that pustule—canker is dis- tinct from IBN. Hickey at V.P.I. (personal correspond— ence) has no evidence that IBN is related to a latent virus. Shelton, et. a1. (33), in North Carolina, working with 5“ Mn, found that it did accumulate in the necrotic areas of the bark tissues. Eggert and Hayden (18) at Purdue have shown,with a modified histochemical technique that manganese does have a relationship to IBN of apples and Was accumulated in the necrotic areas of the bark tissue. Cahoon and Banta (10), at Wooster, Ohio, stated that more than one factor may be involved with IBN. They believed that the disorder was associated with the total accumulation of heavy metals and believed aluminum to be directly involved. Forshey (20) stated that it was an interference with the normal nitrogen metabolism of the plant with an accumulation of toxic nitrogenous intermediates that was responsible for the characteristic , damage to the tissue. Eggert et. a1. (19) believed that the accumulation of toxic amounts of manganese in the tissue could be responsible for the death of the tissue. Fucik's (21) research with apple trees grown in water culture with varying levels of manganese and cal~ cium, indicated that calcium was an important regulator in the absorption of manganese. Many investigators have linked the occurrence of IBN with low soil pH and soils high in manganese (8, 3, 23, 31). Hildebrande (24) found that one ton of high calcium lime raised the pH from 4.0 to 5.0 and with this the leaves of the affected trees regained normal leaf characteristics. Clulo (3) found that calcium hydroxide, calcium carbonate, magnesium carbonate, magnesium oxide, and sodium carbonate all reduced the development of the disorder. Other researchers (8,20) report the use of lime as the common correction for IBN. EXPERIMENTS Experiment I In the summer of 1967, an experiment was conducted to determine whether heavy metals and/or deficient boron would induce the disorder of internal bark necrosis (IBN) in young Delicious apple trees. Two—year—old nursery trees of the variety Miller Sturdy Spur Delicious budded on E.M. VII were planted in three—gallon plastic pails in clean washed quartz sand. Approximately one—third of the roots and tops of the trees was removed from the trees before planting in the small container. The trees were placed on a concrete apron at the Horticultural Research Center. The containers were fitted for automatic watering using the Chapin ring system.1 Pumps were used to deliver a measured amount of solution from storage drums. The trees were watered each day using nutrient solutions. The amount of solution applied at each watering was approximately 1,000 m1. This amount of solution proved sufficient to leach the containers and retard salt accumulation. lChapin Watermatic Company. New York. 10 The experimental design was a randomized block with two single-tree replicates. Normal Ca trees were watered on alternate days with a complete one-half Hoagland2 nutrient solution. Trees receiving the minus B treatment were watered on alternate days with a one—half Hoagland solution minus B. The trees receiving the low Ca treatment were watered on a1- ternate days with a one—half Hoagland solution contain- ing one—sixth of the normal concentration of Ca. Nutri- ent solutions were adjusted to a pH of 4.5. Separate solutions with pH adjusted to 4.5 were made and applied manually to the trees on alternate days. These solutions provided the following treatments: 100 ppm Mn, 200 ppm Mn, 100 ppm Fe, 200 ppm Fe, 100 ppm Cu, 200 ppm Cu, 100 ppm Al, and a solution of 50 ppm each Mn, Fe, Cu and A1. Thus the final array of treatments was for minus boron trees plus the applied treatment with two single tree replicates as follows: 100 ppm 200 ppm Low Ca (53.3 ppm) Mn Fe Cu Al Mn Fe Cu A1 MEM* Normal Ca (160 ppm) Mn Fe Cu A1 Mn Fe Cu A1 MEM *MEM — minor element mix containing 50 ppm each of Mn, Fe, Cu and Al. ' In (October) 1967, the trees were placed in cold storage at 35° F and were stored for five months to O CD. R. Hoagland and D. I. Arnon. 1950. The water— culture method for growing plants without soil. Calif. Agr. Exp. Cir. 347. ll satisfy the rest requirement of the trees. On February 23, 1968, the trees were removed from storage and placed on reenhouse benches in the Plant Science Greenhouses. All fertilization treatments were then continued. Leaf samples, from the middle of the terminal growth, were taken in August of 1967 and again in May of 1968. Bark samples were taken from the old (1967) and new bark (1968) in May of 1968. These samples were ana— lyzed for Ca, Mg, Mn, Fe, Ca, B, Al nutrient composition in the plant Analysis Laboratory by spectrographic ana— lysis. Results From visual observation made in 1967, only the 100 and 200 ppm Mn and minus B treatments developed symptoms of IBN. All but two of the trees treated with 100 and 200 ppm Mn developed IBN symptoms. 0f the four trees that received minus B, three of them developed IBN symp— toms. After the trees had received treatments for three months in the greenhouse, the incidence of IBN was re- corded. It was found that all trees receiving 100 and 200 ppm Mn and minus B had symptoms of IBN. The Mn treatments (Figure 1C) developed more severe symptoms of IBN as denoted by the numerous raised necrotic areas (pimples) on the bark, than the minus B (Figure 1A) which had a sparse distribution of pimples. 12 The results of spectrographic analysis of leaf sam— ples taken in 1967 and 1968, and the results of bark sam- ples of old (1967) and‘new (1968) bark, Table 1, showed the Mn content of the leaf and bark tissues increased significantly over the controls for the 100 and 200 ppm Mn treatments. Leaf Mn was significantly higher in 1968 than in 1967, and the low Ca level, an average for two years,(1eaf 881 and bark 589 ppm), was significantly higher in Mn than the normal Ca level, (leaf 677 and bark 476 ppm). With the leaf Mn values, there was a significant years x Ca level and years x treatment inter— action. The year x Ca level interaction was denoted by the small change in Mn content for 1967; average for low Ca level 437 ppm, average for normal Ca level 380 ppm; compared to the large change in Mn with Ca level for 1968; average for low Ca level 1,325 ppm; average for normal Ca level 964 ppm. The years x treatment inter— action, Table 1, showed that there was a greater increase of Mn with treatment for 1968 than there was for 1967. The results, Table 2, showed the leaf and bark tis— sues of the minus B treatment to be significantly lower in B than the control tissues. Little difference was found in B content within treatments with regard to bark age. A very pronounced rossette condition and stunting was observed with the 200 ppm Fe treatment (Figure 1B). 13 TABLE l.——Mn content of leaf and bark tissue as affected by varying concentrations of Mn and Ca. Low Ca Normal Ca Average (53.3 ppm) (160 ppm) Treatment 1967 1968 1967 1968 1967 1968 Control 100 ppm 200 ppm 50 ppm Average Control 100 ppm 200 ppm 50 ppm Average Mn Mn Mn Mn Mn Mn Leaf Manganese ppm 95 141 73 82 84 112 719** 2161** 539* 1445** 629** 1803** 585** 2065** 599** 1673** 592** 1869** 350 933** 351 658* 350 795** 437 1325 390 964 414a 1145 Bark Manganese ppm 28 60 28 43 28 51 1010** 1024** 590** 1235** 800** 869** 1013** 1096** 750** 714** 881** 1165** 288 196 209 244 248 220 559 595 394 585 489 576 * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. a 1967 Mn means significantly different from 1968 at 1% level. 1 Mix containing 50 ppm Mn, Fe, Cu and Al. TABLE 2.-—B content of leaf and bark tissue as affected by an absence of B from the nutrient solution. Leaf B ppm Treatment 1967 1968 Average Control 23.7 43.1 33.4 Minus Boron 5.4** 7.1** 6.7** ** Means significantly different from control at 1% level. 1,, Figure 1 14 Delicious trees in second year of heavy metal and minus B treatments. A. B. *IJLTJ A tree from minus B treatment. IBN severe. A tree from high (200 ppm) Fe. No IBN, pronounced rosetting. A tree from high (200 ppm) Mn. IBN severe. A tree from high (200 ppm) Cu. No IBN, very short terminal growth. A tree from high (200 ppm) A1. No IBN. A tree from the mix (50 ppm each of Mn, Cu, Fe and Al). No IBN. 15 16 These symptoms were very similar to those described by Shannon (32) to be Fe toxicity. The leaf Fe values (Table 3) increased significantly for 1968 over 1967. There was no significant difference between Ca levels for Fe, low Ca 893 ppm; normal Ca 562 ppm. The average Fe value showed a significant increase for the 200 ppm treatment over the control. TABLE 3.——Fe content of leaf and bark tissue as affected by varying concentrations of Fe and Ca. Low Ca Normal Ca Average (53.3ppm) (160 ppm) "— Treatment 1967 1968 1967 1968 1967 1968 Leaf Iron ppm Control 274 333 168 579 221 456 100 ppm Fe 825* 1,020* 601 858 713 939 200 ppm Fel 914* 1,568** 722* 1,025 818* 1,296* 50 ppm Fe.) 604 1,259** 386 687 495 873 Average 654 1,000 469 787 562a 893 Bark Iron ppm Control 53 56 58 71 55 63 100 ppm Fe 94 160** 103 112 98 136* 200 ppm Fel 136** 183** 106 121 121* 152** 50 ppm Fe) 136** 230** 91 172** 113* 201** Average 105 157 89 119 97a 138 * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. a 1967 Fe means significantly different from 1968 at 1% level. 1) Mix containing 50 ppm Mn, Fe, Ca and Al. Bark analysis (Table 3) for the Fe treatments showed a significant increase in Fe content in 1968 over 1967 and 17 and low Ca,an average for two years 131 ppm, over normal Ca, 104 ppm. With the average Fe values for bark, all but the 100 ppm treatment in 1967 were significantly dif— ferent from the control. V A symptom of Cu toxicity is shown by the 200 ppm Cu treatment (Figure 1D). The symptom was expressed as short terminal growth and stunting of the foliage of the tree. Some trees showed terminal die—back. Leaf Cu values (Table 4) in 1968 were significantly greater than in 1967. No significant difference was ob— served for Ca level, average value for two years, low Ca 62.3 ppm and normal Ca 53.5 ppm. For the average treat- ment values of Cu in 1967 and 1968, all but the 1967 100 ppm treatmentweresignificantly different from the control. There was a significant year x treatment inter— action because of the small change in Cu for the control and 100 ppm treatment partly due to the control receiv— ing a smaller amount of Cu, versus the larger accumula— tion for the 50 and 200 ppm treatment. Bark analysis for Cu (Table 4) showed a significant increase in Cu for 1968 over 1967 and no significant dif— ference between Ca levels, an average for two years, low Ca 65.8 ppm and normal Ca 58.5 ppm. With the average treatment Cu values for 1967 and 1968, all but the 100 ppm Cu treatment in 1967 showed a significant increase over the control. 18 TABLE 4.-—Cu content of leaf and bark tissue as affected by varying concentrations of Cu and Ca. Low Ca Normal Ca Average (53.3 ppm) (150 ppml Treatment 1967 1968 1967 1968 1967 1968 Leaf Copper ppm Control 11.4 19.1 10.7 19.0 11.1 19.1 100 ppm Cu 32.9 59.1* 23.1 56.1* 28.0 57.6* 200 ppm Cul 32.2 106.2** 58.0* 104.8** 45.1* 105.5** 50 ppm Cu 47.0* 120.1** 110.0**117.0** 78.6**118.0** Average 30.9 76.1 50.5 74.2 40.7a 75.1 Bark Copper ppm Control 22.1 30.5 23.5 30.4 22.8 30.5 100 ppm Cu 55.4* 74.2** 38.0 58.8* 46.7 66.5** 200 ppm Cul 58.1** 74.4** 58.2** 74.3** 58.2** 74.3** 50 ppm 09,} 64.0**147.4** 88.7** 96.0** 76.4**121.7** Average 52.4 81.6 49.9 64.9 51.0a 73.3 * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. a 1967 Cu means significantly different from 1968 at 1% level. 1) Mix containing 50 ppm Mn, Fe, Ca and Al. There was a significant year x Ca level x treatment interaction. The treatment x Ca level interaction was the result of the Cu content of 200 ppm Cu being higher than 100 ppm for normal Ca, but not for low Ca. The interaction of years x treatments resulted from a greater increase in Cu for the treatments over the controls for 1968; and the inter— action with Ca levels x years seen by the average value of Cu for all treatments for low and normal Ca being much closer in 1967, low Ca 52.4 ppm and normal Ca 49.9 ppm, than in 1968, low Ca 81.6 ppm and normal Ca 64.9 ppm. 19 The 200 ppm A1 treatment (FigurelE)showed a delayed foliation of the tree. The terminal bud would break first, and then the lateral buds would leaf—out one to three days later. The symptom looked very similar to that of a tree that had not received adequate cold to satisfy the rest requirement. The leaf A1 values (Table 5) showed a significant increase in A1 for 1968 over 1967. There was no signifi— cant difference between Ca levels, average values for two years, low Ca 422.4 ppm and normal Ca 377.7 ppm. The 200 ppm treatment was significantly different from the control in all cases; and the 100 ppm treatment was significantly different from the control in 1968. The bark analysis (Table 5) for Al was not signifi— cantly influenced by treatment. The mix treatment, which was a mixture of 50 ppm each of Mn, Fe, Cu and A1, had symptoms very similar to those of the copper treatment (Figurelfifi, These symptoms would be expected after observing the Cu values for the mix treatment in Table 4. Discussion In the experiment where heavy metals and minus B were used to try to induce IBN, IBN occurred with only 100 and 200 ppm Mn and minus B treatments. That IBN was associated with high leaf value of 500 to 2,000 ppm manganese agreed with the research 20 reported by Zeiders et. a1. (39), Berg et. a1. (8) and others (1, 2, 6, 7, 13, 14, 15, 19, 20, 21, 24, 26, 28, 31, 33). ' TABLE 5.——A1 content of leaf and bark tissue as affected by varying concentrations of A1 and Ca. Low Ca Normal Ca Average (53.3 ppm) (160 ppm) Treatment 1967 1968 1967 1968 1967 1968 Leaf A1 ppm Control 177 325 151 328 164 327 100 ppm A1 277 666** 322 549* 299 608** 200 ppm All 486* 840** 360* 685** 423** 762** 50 ppm A1/) 322 284 233 391 278 338 Average 316 529 267 489 291.3a 508.9 Bark Al ppm Control 51 63 51 50 51 56 100 ppm Al 89 64 88 74 89 69 200 ppm All 63 7O 64 76 64 73 50 ppm 41) 51 64 63 53 57 58 Average 64 65 66 64 65 64 N S N S N S N.S N S N S N.S. No significant difference between means. * Means significantly different from control at 5% level. ** Means sginificantly different from control at 1% level. a 1967 A1 mean significantly different from 1968 at 1% level. 1) Mix containing 50 ppm Mn, Fe, Ca and A1. Low B values (5 to 7 ppm B) were associated with IBN agreed with the research of Zeiders et. a1. (39), Young et. a1. (37), Shannon (32) and Hildebrand (23), but Berg et. a1. (8) and other researchers (6, 31, 34) were 21 not able to find an association between low B and IBN. This relationship may not exist if the trees in their studies had not depleted the residual boron supply in various tissues of the tree. Berg et. a1. (8) showed Mn values to be higher in leaf than in bark tissue, which agreed with the results of this experiment. Nagai et. a1. (26) reported Mn con— tent to be higher in two to three—year old bark than in current season's bark. However, in this experiment current season growth was found to be higher in Mn than the older bark. The high 100 and 200 ppm Fe treatments gave typical rosetting and stunting of the tree as reported by Shannon (32) and Zeiders et. a1. (39). But no IBN was observed on the trees as reported by Shannon (32). The root symp— toms of dark roots for the 100 and 200 ppm Fe treatments were the same as reported by Zeiders (39), and this may be a result of the sequestrene form of Fe used. The mix treatment showed near toxic concentrations of Mn, Fe and Cu but therewere no visible or external symptoms of IBN. This evidence did not support the work of Cahoon et. a1. (10) or Forshey (20) who reported that the disorder could be caused by an accumulation of heavy metals. Zeiders et. a1. (39), stated that high levels of Fe would delay, but not inhibit the expression of the symptoms of IBN. This, in part, was seen by the mix not 22 showing IBN where a high Mn value (Table l; 50 ppm treat- ment low Ca 1968) and high Fe value (Table 3; 50 ppm treatment low Ca 1968) were observed. ' With leaf values, only Mn was found to be increased with the low level of Ca. Fucik (21) has reported that Ca did have an effect on the uptake of Mn. With bark values, the low Ca level had higher values for Mn and Fe; and Cu and Al values were not significantly affected by Ca level. With regard to bark, Mn, Fe and Cu were found in greater concentrations in the new bark (1968). Al, un- like the other elements, was not found to accumulate in bark tissue even though significantly different accumu— lations of A1 with amount applied were observed in the leaf. This suggests that Al was not held in the bark tissue and thus precluded the possibility of Al excess causing IBN. From this experiment it was concluded that IBN could be the result of excess Mn or deficient B, and that it was not associated with excess amounts of Fe, Cu or A1. Experiment 11 In the summer of 1968, an experiment was designed to determine the minimum level of Mn that would induce IBlI. 23 Miller Sturdy Spur Delicious trees on E.M. VII rootstock were used with potting, nutrient solutions (to supply low and normal Ca), and plot design being the same as for Experiment I. The solutions of Mn (100, 75, 50, 25, and 0 ppm) were applied at the rate of 1,000 ml per- tree every other day, alternating with the Ca solutions. The trees were grown during the summer, 1968, at the Horticulture Research Center and in the fall, 1968, moved into cold storage. The trees were moved to the greenhouse in February, 1969, and the treatments were then resumed. Leaf samples from the center of current season's growth were taken in August of 1968 and again in May of 1969. Nutrient analysis was determined in the Plant Analysis Laboratory by spectographic analysis. The sever— ity of IBN was rated for each tree in September, 1968, and again in May, 1969. The rating is presented in Table 6. TABLE 6.——IBN rating scheme for determining the severity of IBN on apple trees. Number of pimples Rating Present in 3 sq. cm. Age of bark Year H None 2 2 2 H 1 1 mzwmw 3 O '1 (D d. :3‘ s1: :3 U1 More than 5 24 Results The IBN rating (Table 7) was significantly greater for low Ca than normal Ca in 1968. There was no signifi— cant difference with Ca levels in 1969 but there was a significant interaction for Mn treatment x Ca level. This was a result of the high ratings in 1969, of the 25 and 50 ppm Mn for the low Ca level. In 1968 only the 75 and 100 ppm Mn were significantly different from the con— trol for the normal Ca level; while with the low Ca level, the 50, 75 and 100 ppm Mn were significantly different from the control. In 1969, only the 25 ppm Mn level with normal Ca level was not significantly different from the control. TABLE 7.——Average IBN rating for Delicious trees grown with varying levels of Mn and Ca for two seasons. 1968 1969 Treatment Normal Ca Low Ca Normal Ca Low Ca (160 ppm) (53.3 ppm) (160 ppm) (53.3 ppm) IBN Rating Control 1.00 1.00 1.00 1.00 25 ppm Mn 1.25 1.50 1.50 4.50** 50 ppm Mn 1.25 2.50* 2.75* 5.00** 75 ppm Mn 2.25* 2.75** 5.00** 5.00** 100 ppm Mn 2.50* 3.75** 5.00** 5.00** * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. The rating of IBN (Table 8) showed a significant effect of Ca level on the response to different Mn 25 concentrations. The rating for IBN for the low Ca level was significantly higher than the control for the 50, 75 and 100 ppm treatments; however, the rating of IBN for the normal Ca level was only significantly different from the control with the 100 ppm Mn. All leaf Mn values, except the 25 ppm Mn treatment at the normal Ca level, were significantly different from the control. TABLE 8.——Rating of IBN on Delicious trees receiving vary— ing levels of Ca and Mn after one growing season. Normal Ca Low Ca (160 ppm) (53.3 ppm) Treatment Rating IBN Mn ppm Rating IBN Mn ppm Control 1.00 80.8 1.00 120.8 25 ppm Mn 1.25 191.0 1.50 500.1** 50 ppm Mn 1.25 329.0* 2.50* 703.0** 75 ppm Mn 2.25 462.3** 2.75* 996.1** 100 ppm Mn 2.50* 629.3** 3.75** 1,168.0** * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. The 50 ppm Mn treatment for the low Ca level showed an IBN rating of 2.5 and a leaf Mn value of 703 ppm. The normal Ca level required the 100 ppm Mn treatment to give a comparable IBN rating of 2.5 and a Mn leaf value of 629.3 ppm. The rating of IBN for the low Ca level was signifi— cantly greater than the normal Ca level in 1968 (Table 9). In 1969, the low Ca level did not significantly increase the rating of IBN above the normal Ca level. The leaf 26 concentration of Ca followed the same pattern as the rat— ing of IBN with the low Ca level being significantly less than the normal Ca level in 1968, but it was not signifi— cantly different in 1969. The leaf concentration of Mn was significantly greater with the low Ca level in both 1968 and 1969. TABLE 9.——Leaf concentration of Ca, Mn and rating of IBN as affected by normal and low Ca levels. Calcium IBN Average Ca Mn level Rating % ppm 19_6§ Normal (160 ppm) 1.55 1.05 337 Low (53.3 ppm) 2 25* .83* 549** 1969 Normal (160 ppm) 3.05 1.13 559 Low (53.3 ppm) 4.10 1.39 673** * Means significantly different at 5% level. ** Means significantly different at 1% level. Figure 2 shows the IBN rating had a positive linear relationship with leaf Mn. The correlation coefficient was 0.5791 and was significant at the .0005 level. The standard error of estimate was 1177.5 ppm Mn. Figure 3 shows that both low and normal Ca were significantly greater than the control in severity of IBN at all dates. The IBN rating for low Ca increased signi— ficantly from October to March while the normal Ca in- creased only slightly for this period. The rating of IBN 27 .mpcoEpmopp :2 mo Loom oco poems em>pomno weapon zmH spas empoaoppoo mocha mSOHoHHoQ mo pcopcoo :2 wood .m onsmwm 28 mNPF F coop _ mhm _ Omh _ Ema mNO _ c<< com - mum _ 0mm — N BUNCH N8| V 29 .ocsw one 30pm: .noQOpoo CH eo>pomno Hoppcoo Ucm do HwEhoc “mo 30H pom zmH mo weapon owmpo>< .m ohsmflm 30 00 mass 0 m:_.om mo o.on 00 sub—:2 we 0 .oao.u0 » m_o..:ou 0U _0E.oZ DU >>od fiuuou Nal 31 for normal Ca increased significantly from March to-June, but the low Ca increased only slightly and this may have been due to the trees receiving low Ca having a maximum rating for IBN in March. The controls were rated one. for all rating dates. Discussion The results of the different Mn concentrations showed that the severity of IBN increased with increasing levels of Mn. However, a given severity rating could have a wide range (150 to 350 ppm) of Mn correlated with it. The low Ca level resulted in a much greater quan- tity of Mn being absorbed in a shorter period of time than the normal Ca level. Fucik (21) showed this rela— tionship. This increased uptake of Mn was evident by the more severe IBN rating given to trees grown with the low Ca solution. Lower levels of Mn applied induced a more severe IBN on trees receiving the low Ca solution. IBN increased while the trees were in storage. This may explain why the growers have observed a severe condition of IBN in the spring after the first growing season for young trees. These results suggest that the substance that caused tissue death or necrosis was also active during the storage period. A leaf composition value of approximately 500 ppm Mn was necessary before young trees would develop severe 32 IBN and need corrective measures. Tree death occurred at Mn concentrations between 800 and 1,000 ppm as reported by other researchers (8,3). Many trees greatly exceeded' this Mn value, but these trees were, in reality, dead at the time of sampling. The rating of 5 for IBN indicated that the trees would probably die during the next grow— ing season or during the dormant period. The rating system for IBN was thought to be ade— quate when it was devised. However, a rating employing a wider range would not have grouped in one category trees severely affected the first year. The highest IBN rating value was too low to permit evaluation of further IBN development. Because the low Ca level and normal Ca level were closer together in terms of IBN rating for the higher Mn treatments the second year than the first year (Table 7), this suggests a need for an expanded rating system. Experiment III In the summer of 1969, an experiment was designed to determine if spur—type Delicious trees were more suscep— tible to IBN than standard Delicious trees. The plot design was randomized block with eight single—tree repli— cates. Trees of Miller Sturdy Spur Delicious on MM 106 and Red Prince Delicious MM 106 rootstock were root and top pruned and potted in washed quartz sand in three— 33 gallon plastic pails. All trees were placed on the auto— matic watering system with the nutrient solution being one—half strength Hoagland solution with Ca reduced to one—sixth strength applied every other day. Mn concentrations of 50, 100 and 150 ppm were used. Applications were of 1,000 m1 of the Mn solutions with the pH adjusted to 4.5 applied to the trees every other day alternating with the nutrient solution. Leaf samples were taken in August, 1969, and the nutrient composition values were determined as before. The trees were rated in September, 1969, as to the sever- ity of IBN in accordance with the previous rating scheme. Results The results (Table 10) showed no significant dif— ference between the two types of trees on the occurrence of IBN. Terminal growth was significantly greater on the standard trees than on the spur-type trees. TABLE 10.—-Standard versus spur—type Delicious apple trees receiving varying levels of Mn (150, 100 and 50 ppm) on the occurrence of IBN. IBN Average Leaf Mn Terminal Growth Type Tree Rating, ppm in inches Standard 3.67 876 14.0 Spur—type 3.71 889 9.0 N.S. N.S. ** ** Difference between means significant at the 1% level. N.S. No significant difference between means. .3” The results showed (Table 11) the leaf Mn concen— tration and IBN rating significantly increased with the increasing concentrations of the Mn treatments. Both 100 and 200 ppm Mn were significantly different from the 50 ppm treatment for leaf Mn and IBN rating. There was no significant tree type x Mn interaction, and no signi— ficant difference in terminal growth was observed with Mn treatment. TABLE 11.—-IBN rating and Mn leaf concentration of stan— dard and spur—type Delicious apple trees as affected by three concentrations of Mn (150, 100 and 50 ppm). Level of Mn Leaf Mn IBN Average Terminal ppm ppm Rating Growth 50 503 1.87 11.75 100 l,004** 4.37** 11.55 150 1,123** 4.81** 11.30 N.S. N.S. Means not significantly different from each other. ** Means significantly different from 50 ppm at 1% level. Discussion The results showed no difference between standard and spur—type Delicious trees treated with varying Mn levels in regard to the severity of IBN. This differs from field observations made by growers and extension agents, who have reported that the disorder (IBN) occurred to a greater degree on the spur—type Delicious trees. 35 The leaf condentration and severity rating of IBN were found to increase with increasing concentrations of Mn as found in previous experiments. The terminal growth of the standard Delicious trees was significantly greater than the growth of the spur—type Delicious trees. Experiment IV In the spring of 1967 and 1968, new Delicious plantings in Michigan were surveyed to determine the pos— sibility of IBN developing. Observations from the pre— vious summer showed that it was almost impossible to deter— mine in older orchards whether the trees were just begin— ning to develop the measled condition, were in the measled condition, or had grown out of the disorder with only the symptoms and not the condition remaining. The only positive way to determine the current stage of IBN was by determining the nutrient content of leaves from the trees. Letters were mailed to different nuseryman in Michigan asking for the name and address of growers in Michigan who had purchased 100 or more Delicious trees for planting in 1967 or 1968. The growers were then con— tacted and leaf samples taken from each of the newly planted orchards. Leaf samples were obtained from many nursery blocks of young Delicious trees. Nutrient anal— ysis was conducted for each sample in the Plant Analysis Laboratory. 35. Manganese and boron compostion values were used to determine if the young trees would develop IBN. From these analyses three orchards were selected in southwest Michigan as being suitable for study of corrective mea— sures for IBN. In the two orchards where the existing trees were used, one orchard was of the variety Miller Sturdy Spur on seedling rootstock, and the other orchard was Miller Sturdy Spur on E.M. VII rootstock. Existing trees were not used in the third orchard because the trees were four to five years old. Therefore, young Delicious trees on seedling rootstocks were planted for this experiment. In the orchards where existing trees were used, the solutions of 50, 30, 10 and 5 pounds of hydrated dolomitic lime (composition Mg, 20.5% and Ca, 37.5%) per 100 gallons of water were applied at the rate of l, 2 or 3 gallons per tree. The solution was applied at the base of the trees where a shallow basin, one and one—half feet in diameter and three to four inches deep, was constructed. The 0 concentration (control) did not receive any solution. The experimental design was randomized block with three two—tree replicates. Also included in these two orchards was an experi— ment designed to determine if the source or amount of ni— trogen applied to the trees would enhance the occurrence of IBN. The nitrogen sources were ammonium nitrate, 37 sodium nitrate and ammonium sulfate applied at rates of one pound and one—half pound of actual nitrogen per tree. The control trees received no nitrogen. The plot design was a randomized block with three two—tree replicates. In the third orchard, where high concentrations of manganese were found in the leaves of the older trees, young Delicious trees on seedling rootstock were planted at a spacing of five feet between trees with the row of young trees planted between two existing tree rows. After planting, the lime solutions, as stated before, were applied into the planting holes after filling. The experimental design was randomized block with three one— tree replicates. Soil samples were taken six inches from the base of the tree and to a depth of six inches at all locations in the fall of 1969. Complete soil analysis, including available Mn, was determined by the Soil Testing Labora— tory at Michigan State University. Leaf samples were taken from the test plots in August of 1969 and complete nutrient analysis determined. The severity of IBN was observed in the fall of 1969 in accordance with the previous rating system. With each of three orchards, the grower was asked to carry out his normal spray program and other cultural practices. 38 Results The survey data showed that most of the young plant— ings sampled had a leaf Mn content between 0 and 200 ppm (Table 12). Only 2 of the orchards sampled had manganese values above 500 ppm. TABLE 12.——Mn content of leaves from newly—planted young Delicious trees (samples the year of planting). Number of Orchards ppm Mn in leaves 1967 1968 Total % of Total 0— 99 ll 5 16 21.05 100—199 18 27 45 59.21 200—299 4 4 8 10.53 300—399 3 O 3 3.94 400—499 1 1 2 2.63 500—599 1 0 1 1.32 600 or more 1 0 l 1.32 Total 39 37 76 100.00 Table 13 shows the manganese content of leaves taken from young Delicious trees budded and growing in Michigan Inusery rows. Most of the trees had a manganese content in the range of 100—199 ppm. This has been reported as the normal manganese concentration range for Delicious apple trees (12). No significant location x treatment interactions were observed for the two sites where existing trees were used, therefore, the leaf and soil analysis results are presented as an average of the two sites. '39 TABLE 13.——Mn leaf content of young Delicious tre nursery row. es in Mn ppm in leaves Rows Samples % of Total 100-199 21 87.50 200—299 2 8.33 300—399 1 4.17 Total 24 100.00 The leaf composition values for Ca, Mg and Mn (Table 14) showed no significant difference from the con— trol with increasing lime concentration. The lea values were seen to decline with increasing lime tration; however, the leaf Mn value was much lowe the control than the 350 to 524 ppm found when th were first sampled in 1967. No significant diffe was found between treatments on the severity of I which was considerably less than in 1967. f Mn concen— r for e trees rence BN, TABLE l4.-—Leaf analysis values and IBN rating of Deli— cious trees treated with lime slurry solutions. Existing orchards. Lb. of hydrated dolomitic 19693 lime/100 gal. H20 Ca% Mg% Mn ppm IBN 0 1.60 .30 174 1.17 5 1.62 .31 173 1.02 10 1.53 .29 154 1.33 30 1.59 .30 149 1.34 50 1.53 .30 154 1.34 N. S. N.S. N.S. N.S. N.S. Means not significantly different from 0 level. a Average of two sites. 40 Soil Mg and pH were found to be significantly in— creased for the 50 lb. lime treatment over the centrol. The other treatments showed no significant increase (Table 15). There was no significant difference between treatments for soil Ca and soil Mn. TABLE 15.——Soil analysis results from Delicious trees treated with lime slurry solutions. Existing orchards. Lb. of hydrated 1969a dolomitic lime/ Soil Ca Soil Mg Soil Mn 100 gal. H2O lb./acre lb./acre ppm Soil pH 0 1,226 335 44.0 5.55 5 1,436 345 48.1 5.68 10 1,522 405 66.6 5.86 30 1,528 540 51.9 6.36 50 1,429 669* 50.0 6.72* a Average of two sites. The rate of application of the lime slurry solution (Table 16) significantly increased the soil pH value for the three—gallon rate. There was no significant increase for any of the other parameters with increasing rates of the lime slurry solution; however, leaf Ca, soil Ca, and soil Mg showed a slight increase with increasing rate of application. The leaf composition values of the planted De— licious trees treated with the lime slurry solutions (Table 17) showed the Ca value not to be significantly 41 increased with increased amounts of lime. The leaf Mg values did increase with increasing concentration of lime, and the highest concentration (50 lbs) was significantly higher than the control. The leaf concentrations of Mn were not significantly different from the control, but there was a slight decline in Mn content for the 5, 10 and 30 1b. treatments. TABLE l6.—-Soil analysis results from Delicious trees treated with the lime slurry solutions. Existing orchards. 1969a Gal. of slurry Ca leaf Mg leaf Ca soil Mg soil Soil per tree % % lb./acre lb./acre pH 1 1.43 .37 1,355 417 5.80 2 1.44 .37 1,301 427 6.10 3 1.49 .37 1,356 450 6.22* * Means significantly different from one-gallon treatment at 5% level. a Average of two sites. TABLE 17.——Leaf analysis values and IBN rating for planted Delicious trees treated with the lime slurry solutions. Newly planted trees. Lb. of dolomitic hydrated lime/ 1969 100 gal. H2O Ca % Mg % Mn ppm IBN 0 1.33 .23 488 3.33 5 1.55 .24 447 2.22 10 1.63 .35 458 2.00 30 1.44 .38 443 2.33 50 1.35 .42* 481 2.44 * Means significantly different from 0 level at 5% level. 42- The IBN rating was less for all levels of the lime concentrations, but none of the means was significantly different. Soil analysis results from the planted Delicious trees (Table 18) showed the soil Ca value increased with higher concentrations of lime. TABLE l8.-—Soil analysis results for planted Delicious trees treated with the lime slurry solutions. Newly planted trees. Lb. of dolomitic 1969 hydrated lime/ Ca Soil Mg Soil Mn Soil 100 gal. H20 lb./acre lb./acre ppm pH 0 555 129 24.7' 6.26 5 692 147 26.7 6.50 10 732 199 24.3 6.95 30 693 368 26.6 7.26* 50 850* 510** 33.1 7.76** * Means significantly different from 0 level at 5% level. ** Means significantly different from 0 level at 1% level. Soil Mg increased with all amounts of liming mater— ial. There was no significant difference between treat— ments with regard to available soil manganese. The soil pH value was raised with increasing concentrations of liming material. Only the 50 1b. concentration was sig— nificantly different from the control (0 treatment) in all cases except soil manganese. The leaf composition values for trees that were treated with three sources of nitrogen and a control are 43 given in Table 19. Leaf value for N, Mn, and the sever— ity of IBN were not significantly different from the con— trol for any source of nitrogen. The Mn value, however, tended to increase for all nitrogen sources. NHuNO3 and (NHu)2SOu had slightly higher values for IBN than the control and the NaNO3 had the lowest rating of any source. TABLE 19.——Leaf composition and IBN rating of trees re— ceiving different sources of N. Existing orchards. 1969a Source N % Mn ppm IBN rating NHMNO3 2.38 208 1.54 (NH4)2SO4 2.38 231 1.67 NaNo3 2 35 217 1.33 Control 2 41 165 1.50 N S N.S. N.S. N.S. Means not significantly different from the control. a Average of two sites. Soil analysis for the trees treated with the nitro— gen sources (Table 20) showed NHuNO and (NHu)2SOu to be 3 significantly higher in soil Mn than the control. Soil acidity was found to be significantly higher where the ammonium nitrate and sulfate were used. The NaNO3 was found to be slightly lower than the control with regard to soil acidity. No significant differences were found with regard to the one pound and one—half pound of actual N rates applied. 44 TABLE 20.——Soil analysis results of trees receiving dif— ference sources of N. 1969a Source Soil Mn ppm pH NHHNO3 97.2* , 4.66** (NHu)280u 78.8* 4.73** NaNO3 52.6 - 5.46 Control 45.2 5.37 * Means significantly different from control at 5% level. ** Means significantly different from control at 1% level. a Average of two sites. Discussion Growers and extension personnel had indicated that IBN was a large problem in Michigan. However, leaf analysis survey data of young Delicious plantings did not suggest IBN to be present in most Michigan plantings. The results showed two orchards out of 76 sampled would probably need corrective lime application for IBN, since a leaf Mn value above 500 ppm was found. The lesser amount of IBN observed could result be— cause the symptoms of IBN remain visible on the bark of the tree after the tree has grown out of the IBN condi— tion. Therefore, orchards may have been reported as hav— ing the disorder when, in fact, the orchard had outgrown the condition even though the bark symptoms were still visible. Sampling in the nursery showed young trees were not accumulating toxic amounts of Mn in the nursery. 45 Thus the trees that did develop IBN were accumulating the Mn after being planted in the growers' orchards. Leaf analysis data showed leaf Ca not to be signi— ficantly different from the controls in the liming ex— periment. With regard to leaf Mg the existing trees did show a significant difference between treatment and the 0 level of lime(control). The leaf Mn value did not increase significantly from the 0 level of lime, but in the nitrogen experiment the leaf Mn value increased slightly with all forms of nigrogen, with (NHu)2SOu hav— ing the highest Mn value. Kenworthy (25) showed the uptake of Mn by cherry trees was greater when NHuNO was 3 applied than when no N was used. IBN rating was not significantly affected by treat- ment; however, the 0 level of lime for the planted Deli— cious tree lime treatments and the NHMNO3 and (NHu)2SOu forms of N had the higher rating for IBN. The soil Mn in the lime experiment was observed to increase slightly with increasing liming material and with decreasing soil acidity. This does not agree with Pailoor's work (27) where he found soil Mn to diminish with decreasing acidity. Also this discrepancy was shown by the fact that the soil Mn values were not in agreement with the leaf Mn content where a slight decrease was seen in leaf Mn with 5, 10 and 30 1b. concentrations of liming solution. One explanation for the higher soil Mn values 46 could be the extracting method used in the soil tests. The method employed was a 0.1 N HCl solution. This solu- tion could possibly extract the easily reducible Mn that was not considered available for uptake by the plant, thereby giving a higher apparent available soil Mn value. The soil Mn and pH were significantly increased for the NHuNO3 and (NHu)2SOM forms of N over the control. Forshey (20) has reported that the ammonium forms of N will increase soil acidity; this increased available Mn. The NaNO3 resulted in a higher soil pH value than the other sources of N. This was in agreement with what has been reported (8) about the failure of NaNO3 to increase soil acidity to the same degree as the ammonium nitrate and sulfate. The two older orchards where lime slurry solutions and nitrogen treatments were applied probably would have grown out of the IBN condition without any corrective measures. Both of these orchards, one showing 524 ppm Mn and the other 350 ppm Mn, were above or just below the critical diagnostic value of 500 ppm Mn. This value had been established from the results of the varying Mn levels experiment and from previous work by Kenworthy to be the value for Mn above which IBN would develop. Also, when no lime or nitrogen was applied, the leaf Mn value decreased to 165—174 ppm in 1969, showing that without treatment of lime, the Mn value decreased below the level that would cause symptoms. 47 Experiment V Investigations were made to determine the location and approximate amounts of elements within the necrotic areas of the diseased tissues. Bark samples were taken from trees treated with excess manganese and low boron that were showing IBN, and a sample was taken from the control trees for comparison. Sample preparation for microprobe analysis differed from that described by Rasmussen (29) in that the sections were cut 15 to 20 microns thick, and then coated in a Varian vacuum evapo- rator with a thin layer of carbon for conductivity. 3 The instrument used was the ARL electron micro— probe X—ray analyzer Model EMX—SM. The instrument con- ditions were 25 KV acceleration voltage and 0.05 micro- amp sample current with the first samples run, and 15 KV acceleration voltage and 0.125 microamp sample current on subsequent samples. The location and amount of the element in question was recorded in two ways. The first way being a line scan across the sample where the X-rays (Kc radiations), that have a characteristic wave length for each element, were recorded on a X,Y recorder. The second way was oscillograms (photographs) prepared from images displayed on the cathode ray tube by either secondary electrons or 3Applied Research Laboratory, Inc., Sunland, Calif. 48 i | Ka radiations detection. The secondary electron oscillograms showed the area being studied, and the X—ray oscillo— grams showed the concentration and location of the element. By use of the line scans and oscillograms, the precise location and semi—quantitative amount of a given element could be determined. Results Line profile analysis of normal (control) apple bark tissue, Figure 4, showed the Mn and B distribution in the tissues to be uniform. The average background count was 34 count/sec, and the average Mn count was 44 count/sec. No unusually large quantitites of Mn were found in any areas of the normal bark tissue. The Mn X—ray oscillogram for the normal bark tissue showed a uniform distribution of Mn for a large area of the nor- mal tissue. Corresponding X—ray oscillogram of Mn and a line profile analysis, Figure 5, showed Mn to accumulate in the necrotic lesion. B, the line scan analyzing for Mn content increased when the necrotic area was analyzed, and the Mn content was found the greatest in the middle of the necrotic area. The background count, line scan B, was uniform through the sample. With use of cryostat sections of bark tissue, the cellular detail of the tissues could be determined, as shown in Figures 6B and 7B. 49 , .Empwoaaflomo mmpux oopnomcfi one so UoBmOHocH mm m on < ucfloo Eopm eommopwopo zoom oCHH one .Empwoaaflomo one so phenom wcflocommonpoo new pmaflsflm m mamsoo nampm one Go opmzow now 620 .CE mo soapsnflppmflo one mcflpmpomcosoo xnmn HmEhoc mo Emnwoaafiomo amplx Ugo mflmzamcm mammopd mead .mpcsoo oczopmxomo owmpo>m one ompmcmflmoo Q .oom\pc:oo OOH was oflmom nomnm Hasm .nom mfimsoo compw one so opmzwm ownma 6:0 .:2 mo demagnflppmfie one mcflpmnpwcoEoo Esme meHOACoo m we mflmzamcm oaamopo mend .m: onswflm .<: opzmflm 50 .13. 1 ,1. 1 ....... ,,,,,,,,, H :1; 1 1 , 1 i.“ _ ,1 .. . . N 1 . . 1 1,. 1 1 . .. 11 ......... 1 _ L ,. a I 11. .1. 1_ 1, 1. 1. 1 | I I .1. . ,1 . ..1.. ,1. T, 11.. 11 ......... ,t}- , 1 ......... . 1. _ .,_ I l .1 . ......... v 1... 1 .. M. _ _ 51 .EwprHHHomo zmplx vmpgmmcfi mzp co Umpmo [HUGH mm m Op < pcfloq Eopm UmmmmLMOLQ cmom mafia mQB .Emhmoaafiomo map so mgmswm wcflccoammppoo Ucw LwHHEHm m mamdvm cgwmw map so mpwswm 10m mco .cz mo COHpsnflppmHU map wcwpwgpmcoEmU xamn zmH mo Ewpwoaaflomo mwglx ccm mflmzamcm mfiflgopm mcflq .m mpswflm 52 '——- "F 5:11- iiiii———-——-h 53 In Figure 6B a triangle designates the center of the necrotic lesion of a high Mn treatment when viewed as’a secondary electron image. The Ca X—ray oscillogram, Figure 6D, corresponding to the same area as the Mn X—ray and secondary electron oscillograms, showed an increased concentration of Ca within the necrotic lesion. Corresponding X—ray oscillo— grams for K, Figure 6C, and P, Figure 6A, showed de— creased concentrations of these elements within the lesion. In Figure 7B, a triangle designates the center of the necrotic lesion induced with the minus B treatment used for analysis. The corresponding Ca X—ray oscillo— gram, Figure 7D, showed an increase of Ca within the necrotic lesion. K and P were shown to decrease within the necrotic lesion as shown by the X—ray oscillograms for K and P, Figures 70 and 7A. Line profile analysis of minus B apple bark tissue, Figure MA, line scan C, showed the Mn content to be uni— formly distributed within the tissue. The Mn content was low as shown by the Mn count, averaging approximately 19 count/sec. on a 100 count/sec. scale; and the average background count, line scan D, being 8 count/sec. Discussion Determination of the Mn content in the necrotic lesions of trees treated with Mn was done with the use 5H Emsmoafiflowo zwplx do . Emhwoaaflomo zmhlx x . EmMmOHHHomo coppooao zpmocoomm thwoaaflomo amplx m