Milton Schroth, Bill Olson, Art McCain, Joe Osgood, Mavis Date-Chong Hendson, Yung-An Lee, and Beth Teviotdale.

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1 NEW APPROACHES TO CONTROL WALNUT BLIGHT Milton Schroth, Bill Olson, Art McCain, Joe Osgood, Mavis Date-Chong Hendson, Yung-An Lee, and Beth Teviotdale. ABSTRACT: The addition of iron chloride to fixed coppers greatly affects the susceptibility of blight bacteria to the toxic action of copper. In addition, by lowering the ph at the leaf and nut surfaces, iron causes much greater amounts of free copper ions to become released from the fixed form, thus enabling them to kill bacteria. Copper and copper-iron mixtures were tested for the second year in the field. The copper-iron mixture. reduced the number of blighted leaves by 177%. Different forms of iron also were tested. The combination of iron chloride with the insoluble iron oxide caused some phytotoxicity. However, the results suggested that combinations of different irons, because of solubility aspects, could be used to develop improved formulations with fixed coppers for persistence on foliage and for improved toxicity to blight bacteria. Data also are presented showing the extent that iron increases the release of free copper ions from fixed coppers. This is shown in water solutions and on the leaf surfaces. Other ~ield research suggested that a late winter spray could markedly reduce the percentage of buds colonized by blight bacteria. The copper and copper-iron mixtures also were tested for efficacy against nut blight. Although the copper-iron was 120% better than copper alone, the great amount of variability among replications prevented the results from being statistically significant. INTRODUCTION: This year's research focused on field testing iron-copper formulations. Previous laboratory and greenhouse data showed that the iron increased the susceptibility of blight bacteria to the toxic action of copper. Moreover, the addition of ferric chloride to fixed coppers lowers the ph, thus increasing the amount of free copper ions. However, this has not been tested on plant.surfaces, an objective of this year's research. Another added benefit of the iron is that. the iron-copper combination kills copper tolerant bacteria whereas the normal fixed coppers do not. As with any new product it will take several years before the optimal formulation can be worked-out. As is, we do not know how much ferric chloride to add to the fixed coppers to obtain greatest efficacy without also increasing the amount of phytotoxicity. In addition, a number of growers have been using ferric sulfate as an amendment to coppers. This needs to be tested experimentally as it is much more insoluble than ferric chloride and may not produce the desired results. It is possible that combinations of the two forms of iron may ultimately prove to be the. best material. OBJECTIVES: 1. Test different forms.of iron to add to fixed coppers in order to improve the control of walnut blight. Determine the role of iron in lowering the ph at the leaf and nutlet surfaces which in turn increases the amount of free copper ~

2 2. Determine by use of a copper electrode the best concentrations of copper and iron to use in the field in relation to persistence and the availability of the free copper ion on walnut foliage. 3. Monitor the populations of blight bacteria throughout the year to determine when best to apply bactericides--fall, winter, and prior to bud break. 4. Investigate the significance of copper tolerance in blight bacteria with respect to their persistence and infectivity. PROCEDURES: 1. Forms of iron--there are many different forms of iron and they differ greatly in solubility. Ferric chloride is very soluble, ferric sulfate is slightly soluble, and iron oxide is very insoluble in water but becomes slowly soluble when on plant surfaces. Much work needs to be done testing these different forms and combinations thereof. This year we field tested ferric chloride and a combination of ferric chloride with ferric oxide. This was done by spraying trees at normal intervals and then evaluating nuts and leaves for blight lesions. The normal fixed coppers were used as standards. 2. Effect of iron in increasing the amount of free copper ions on leaves-- This was done using English walnut seedlings grown in the greenhouse. Leaves were sprayed with standard concentrations of fixed copper or fixed copper.plus ferric chloride. The leaves were allowed to dry. Twenty-fourhours later, the leaves were moistened by a light water spray, the droplets on leaves were collected, and the amount of free copper i?ns analyzed with a copper electrode. This provides information about the availability of copper ions to kill blight bacteria. 3. Do fall, winter, or early spring sprays reduce the incidence of buds infested with blight bacteria?--this was tested by spraying trees at different times with fixed coppers and then monitoring large numbers of buds for the incidence of blight bacteria. The process calls for the sampling of hundreds of individual buds on special media in the laboratory. The colonies of blight bacteria are" then easily recognized.thereby enabling an evaluation of the number of buds that have been colonized by the bacteria. The trees were sprayed in November and again in late January or early February. Some trees were treated only in late January or early February. The buds were sampled j~st before bud break. 4. Incidence of copper tolerant blight bacteria--this work was not done in 1991 because of the amount of effort devoted to the other objectives. RESULTS AND CONCLUSIONS: This year's research shows conclusively that the addition of iron to copper greatly increases its efficacy. The best data were obtained by evaluating leaf blight. Table 1 summarizes data from two field plots and indicates that a 177% reduction in percentage of blighted leaves occurred when comparing iron-copper to copper alone. This was statistically significant at P-O.OS. As exp~cted, both the iron-copper and copper (Champion) substantially reduced leaf blight compared to the control. This demonstrated the superiority of iron-copper to copper alone

3 Table 1. Percentage of walnut leaflets infected with Xa..n.tb.Am.a.n campestris pv. ~ on walnut trees with different treatments. Treatmentswx first surveyyz second survey Champion 9.5a 12.2a Champion+FeCla 6.6a 4.4b Champion+FeCla+Fe20a 5.6a 5.5b Control 19.7b 27.4c w100 ppm Fe3+ of FeCI3 were applied In the second treatment (Champlon+FeCI3). 50 ppm Fe3+ of FeCI3 and Fe203 (total 100 ppm Fe3+) were applied In the third treatment (Champion+FeCI3+Fe203)' xchampion were applied at the rate of 2 Ibl100ga!. All treatments were applied to the Ashley variety, beginning on 16 March1991, and sprayed 6 times at intervals of days. First samples were collected on 18 April 1991, and second samples wer.e collected on 10 June YValues are the mean number for each treatment replicated six times In completely randomized design leaflet for each replicate were assayed for the Infection of X. C.pv juolandis.. zmeans with a column followed by the same letter are not significantly different (P- 0.05), least significant diff~rent. 4.6 (first survey) and 6.0 (second survey). The mixture of ferric chloride with insoluble iron oxide resulted in very interesting findings. Although the amount of blight was reduced to approximately the same extent of that obtained with iron-copper, phytotoxicity was noted. This was surprising in that the iron oxide is relatively insoluble. Possibly the iron oxide remained on the leaves (resisting washing-off by rainfall) and the slow release of copper resul t:ed in internal copper accumulation by the leaves. On the other hand, the amount of free copper ions on the leaf surfaces immediately after spraying was half since only half the amount of ferric chloride was used. These peculiar and unexpected results suggest that there are great opportunities to make various kinds of iron-copper formulations which differ in persistence, phytotoxicity, amount of free copper ions, etc. Ideally, the optimum formulation will be one that has the greatest amount of free copper ions available to kill blight bacteria and one that is not easily washed-off leaves during rain fall. In addition, there should be a sl~w release of free copper ions to make-up for those that become bound to plant surfaces and plant exudates. Lastly, there should be low to no phytotoxicity. Potency of free copper ions on leaf surfaces. Aside from the fact that iron somehowaltersthe physiology of. blight bacteria causing them to become more susceptible to the toxi.c acti~n of the copper ion, ferric chloride greatly increases the amount of ~ree copper ions on leaf surfaces. By use of the copper electrode,water drops were c'ollected from leaves that were sprayed with copper alone and copper with varying amounts of iron. As shown in table 2, iron caused a marked increase in the amount of free copper ions. The data are presented two 282

4 _ ways: one column shows the release of free copper from copper hydroxide in a water solution whereas another column shows the amount released on walnut leaf surfaces. As can be seen, the extent of free copper ions increased with added concentrations of iron. Thus, this again shows the importance of doing extensive field testing with different combinations of iron and copper to determine which formulation is best to control blight. Table 2. ph values and free copper ion concentrationsof Kocidesolutionsand walnut leaf surfacessprayed with Kocidesolutions amendedwith different concentrationsof iron. ============================================================ Kocide (11b/SO gal) water solutions on leaf surfaces Added Fe3+ (ppm) ph [Cu2+] ppm ph [Cu2+] ppm o % % % :i: % :i: % :i: % :i: What about dormant sprays to reduce blight bacteria? It is difficult to determine the importance of dormant sprays by relying solely on nut blight data. This, in part, is because nutlets drop-off. An alternative method is to determine if dormant sprays reduced the amount of inoculum. This is done by assaying individual buds for populations of the bacteria after spray treatments. The rationale is that the less bacteria present, the less amount of blight may occur. However, this may not be true if weather, conducive for multiplication of bl ight bacteria, persists 'for long periods of time. In these cases, multiplication of the bacteria can be so fast that the amount of inoculum is not of such importance. The field test (table 3) showed that late winter sprays substantially reduced the number of buds infested with blight bacteria well over 100X. The key factor seemed to be the time of application. The November spray had no affect on the incidence of infested buds as evaluated in ~arch. However, the mid-january and February sprays markedly reduced the amount of blight bacteria. These results were unexpected; the experimentation needs to be repeated in succeeding years for purposes of validation. 283

5 Table 3. Percentageof walnutbuds infestedwith~ campestris pv. l.um.andii.on walnut trees sprayed with Kocide 101 at different time. Date of sprayingx percentage (%)yz /7/90-1/17/91 2/14/91 11/7/90 + 1/1 7/91 + 1/17/91 2/14/ a 22.0bc 28.5ac 24.8bc 20.3bc xkocide 101 were applied at the rate of 2 Ibl100gai. All treatments were applied to the Ashley variety. The samples were colledted on 1 March YValuesare the mean number for each treatment replicated four times in completely randomized design. 60 buds for each treatment were assayed for the infestion of X.,-. pv juolandis. zmeans with a column followed by the same letter are not significantly different" (P ), least significant different Effect of iron in reducini nut bliiht: A major effort was made to determine the effect of iron-copper compared to copper alone in red~cing nut blight. This was done by tagging nutlets, counting those that were blighted, and counting t~e number that fell-off trees. Unfortunately, there was great variation among the replications making it impossible to obtain statistically significant results

6 One of the problems was that the experiment included a number of trees that served as controls the previous year. We suspect that the great amount of carryover bacteria contributed to the erratic results. Th~s, even though the ironcopper treatments resulted in a 1201 improvement over the copper alone, results were not significant at the P level (table 4). This year larger plots will be used. Table 4. Percentage of walnut nutlets infected with Xanthomonas campestris pv. ~ on walnut trees with differenttreatments. Treatmentswx percentage (%)Yz Champion 12.3a Champion+FeCl3 5.6a Champion+FeCI3+Fe a Control 34.6b w100 ppm Fe3+ of FeCI3 were applied in the second treatment (Champlon+FeCI3). 50 ppm Fe3+ of FeCI3 and Fe203 (total 100 ppm Fe3+) were applied in the third treatment (Champlon+FeCI3+Fe203)' xchampion were applied at the rate of 2 Ibl100gal. All treatments were applied to the Ashley variety, beginning on 16 March1991, and sprayed 6 times at Intervals of days. Nutlets were surveyed on 10 June YValues are the mean number for each treatment replicated six times In completely randomized design. 80 nutlets for each replicate were assayed for the Infection of X.,. pv luolandis. zmeans with a column f6110wedby the same letter are not significantly different (P- 0.05), least significant different