HORTSCIENCE 53(1):4–8. 2018. https://doi.org/10.21273/HORTSCI12038-17 Unlike most root pathogens, which produce spores that lie dormant in the soil (e.g., Phytophthora), Armillaria persists in its veg- Screening Almond Rootstocks for etative stage—mycelium—within woody roots left in the soil after clearing infected Sources of Resistance to Armillaria trees (residual roots). We have recovered viable A. mellea mycelium from residual Root Disease roots, in orchard and vineyard soil, originat- ing from tree crops and forest trees 10+ years Kendra Baumgartner1 and Phillip Fujiyoshi after they were cleared (Baumgartner and U.S. Department of Agriculture, Agricultural Research Service, Crops Rizzo, 2002; Rizzo et al., 1998). Fumigants Pathology and Genetics Research Unit, Davis, CA 95616 such as methyl bromide are only effective to the extent that they reach and penetrate Craig Ledbetter residual roots (Bliss, 1951; Munnecke et al., U.S. Department of Agriculture, Agricultural Research Service, Crop 1981). Injection of the systemic fungicide propiconazole to living trees reduced symp- Diseases, Pests and Genetics Research Unit, Parlier, CA 93648 tom severity in almond on Lovell rootstock Roger Duncan (Adaskaveg et al., 1999), but this approach can be cumbersome for large disease centers. University of California Cooperative Extension, Stanislaus County, Modesto, Another approach to postinfection manage- CA 94358 ment is root collar excavation, although this approach must be carried out in advance of Daniel A. Kluepfel severe symptom development (Baumgartner, U.S. Department of Agriculture, Agricultural Research Service, Crops 2004; Schnabel et al., 2012). Pathology and Genetics Research Unit, Davis, CA 95616 A 10-year field trial in France of ungrafted Prunus rootstocks (Guillaumin et al., 1989) Additional index words. Armillaria mellea, Armillaria tabescens, clonal rootstocks, Prunus identified A. mellea-resistant and susceptible dulcis, Prunus persica Prunus species. Peach (GF305) and peach · Abstract. Prunus dulcis (almond) is one of the most susceptible horticultural crops to hybrids [S3400 (Prunus besseyi peach) and · Armillaria root disease. Resistance to Armillaria mellea and Armillaria tabescens, the ‘Paramount’, aka GF677 (almond peach)] geographically isolated causal fungi that attack almond and closely related Prunus were most susceptible. By contrast, root- persica (peach), has been evaluated in studies of almond, peach, and other Prunus stocks with plum backgrounds [‘Myrabi’ rootstocks, but not in one comprehensive study. We evaluated the relative resistance to A. (Myrobalan), GF43 (Prunus domestica), · mellea and A. tabescens of six clonally propagated almond and peach rootstocks (Bright’s 5, Marianna GF8-1 (Myrobalan Prunus mun- Empyrean 1, Hansen 536, Krymsk 1, Krymsk 86, and Lovell) in comparison with that of soniana)] were the most resistant. Similar clonally propagated Marianna 2624 rootstock (resistant control) and clonally propa- results of lower mortality among rootstocks gated Nemaguard rootstock (susceptible control). Replicate clones used in the growth with plum parentage including Myrobalan chamber assay were micropropagated and rooted in vitro before inoculating the culture (P. cerasifera), P. munsoniana, or both were medium with Armillaria spp. At 2 months, the most resistant and susceptible rootstocks found in a field trial in California (Thomas were Krymsk 86 and Hansen 536, respectively, with 27% vs. 89% mortality. This finding et al., 1948). Interestingly, in the French was consistent among two isolates of A. mellea and one isolate of A. tabescens in three study, hybrids of peach and Myrobalan · · replicate experiments. Our finding of low mortality among Krymsk 86, Krymsk 1, and {‘Ishtara’ [Myrobalan (Myrobalan peach)], · Marianna 2624, which all share Prunus cerasifera (Myrobalan plum) parentage, is ‘Myran’ (Myrobalan peach)} were resis- consistent with past reports of resistance in the field to A. mellea, but conflicts with tant, suggesting that resistance in Myrobalan reports of susceptibility to A. tabescens. Resistance to A. tabescens of genotypes with is a dominant trait. Mortality was related to Myrobalan plum parentage in our assay may reflect the simplified rooting environment the degree of infection, such that rootstocks of tissue culture medium, which does not perfectly mimic a field trial, in which biotic and with low mortality also had very few infected abiotic factors may affect host resistance. Nonetheless, our growth chamber assay may roots, and a low proportion of plants were provide a more rapid alternative to identify sources of resistance for breeding and to girdled by root collar infection. These find- screen progeny of such crosses. ings suggest that the Armillaria resistance in Myrobalan, and possibly other plums, is true resistance, rather than tolerance. Resistant Prunus species are among the most sus- (Schnabel et al., 2005) and it limits the rootstocks became infected, but infection ceptible tree crops to Armillaria root disease productive lifespan of peach and necessitates was restricted to a low level. If Myrobalan in the northern hemisphere (Baumgartner premature removal and replanting of entire were ‘‘tolerant,’’ we would expect low mor- et al., 2011). In the southeastern United orchards (Schnabel et al., 2012). Outside the tality, in spite of high levels of root infection. States, the causal species is A. tabescens United States, there are similar reports of Field trials examining rootstock resis- stonefruit susceptibility from Mexico (Elias- tance to soil-borne pathogens are informa- Roman et al., 2013), Europe (Guillaumin tive, but they are lengthy and cannot be et al., 1989), the United Kingdom (Kable, modified after planting to accommodate Received for publication 18 Apr. 2017. Accepted 1974), Japan (Sasaki and Jinno, 1975), and new rootstocks. Unfortunately, inconsistent for publication 13 July 2017. China (Qin et al., 2007). The causal fungi infection in the greenhouse has been a bottle- Funding provided by the Almond Board of Cal- across these regions, A. mellea or A. tabes- neck to Armillaria research. Repeated at- ifornia. Tissue culture stocks of rootstocks were cens, colonize and kill woody roots and then tempts by researchers to modify each step provided by Javier Castillon (Duarte Nursery/Dry decompose the root wood as substrate. Such of the greenhouse infection assay have been Creek Laboratories, Hughson, CA). destruction to the root system over time unsuccessful at counteracting the high pro- Review of this manuscript and isolates of Armil- laria tabescens from Guido Schnabel (Plant & reduces yield and diminishes growth capacity portion of plants that escape infection and the Environmental Sciences Department, Clemson (Baumgartner, 2004), eventually killing in- slow rate of infection (Guillaumin et al., University) are gratefully acknowledged. fected trees. 1989; Mansilla et al., 2001; Singh, 1980). 1Corresponding author. E-mail: kendra.baumgartner@ Management of Armillaria root disease Rather than trying to recreate a field infection ars.usda.gov. focuses mainly on preplant soil fumigation. of plants grown in pots in a soil-based medium,

4 HORTSCIENCE VOL. 53(1) JANUARY 2018 | BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES we grew plants in a tissue culture medium, could easily be selected for Armillaria in- percent mortality. ANOVA was performed which supports both plants and pathogen oculation at a later date. using the MIXED procedure in SAS, with (Baumgartner et al., 2010; Baumgartner Three Armillaria isolates (two A. mellea Kenward–Roger as the denominator df et al., 2013). With this infection assay, we and one A. tabescens) were inoculated sepa- method (Littell et al., 1996). All main and have overcome the major barriers of the rately to eight plant genotypes, in each of three interaction effects were considered fixed. F greenhouse approach, namely, eliminating experiments. The two isolates of A. mellea, values with P < 0.05 were considered signif- ‘‘escapes,’’ and bringing about consistent Sac304 and Sol310, were recovered from icant. Before ANOVA, homogeneity of var- and repeatable levels of mortality. We can symptomatic cherry and French prune, re- iance across treatments was confirmed (Box replicate experiments within a 1-year period spectively (Sacramento County, CA, and et al., 1978). After ANOVA, for significant to identify promising rootstock genotypes for Solano County, CA, respectively). Of our effects (P < 0.05), differences among means later, more lengthy evaluation in the field. collection of 16 A. mellea isolates from were assessed based on the presence/absence Our objective was to use this infection Prunus, these two isolates exhibited similar of overlap of their 95% confidence intervals, assay to identify Armillaria-resistant root- growth in culture (data not shown). Also, and means without overlapping intervals stocks for almond, by screening a set of eight exhibiting similar growth in culture was one were considered significantly different clonally propagated, commercially available isolate of A. tabescens, SC4 (aka SC.LD- (Westfall et al., 1999). rootstocks, which are graft-compatible with 1.02), which was recovered from symptomatic almond. Plant genotypes were selected based peach in Greenville County, SC (Schnabel Results on input from University of California Co- et al., 2005). Inoculum was prepared by operative Extension crop advisors and al- homogenizing for 30 s a 7-d culture grown The ranking of relative resistance of mond researchers, with a focus on the in potato dextrose broth (PDB) with 2.5 mM Marianna 2624 as greater than that of Nem- following criteria: widespread use in Califor- sodium acetate (25 C, 100 rpm) and then aguard comes primarily from field observa- nia (Lovell and Nemaguard), building in- transferring with a sterile 1-mL glass pipette tions (Thomas et al., 1948). A preliminary terest for new plantings (peach · almond 200 mL of the resulting homogenate (i.e., experiment was conducted to confirm differ- hybrids Bright’s 5, Empyrean 1, and Hansen mycelial fragments) per plant onto the surface ences in mortality between our clonally prop- 536), or likely to be Armillaria resistant of the plant growth medium (Baumgartner agated Nemaguard and Marianna 2624, (Myrobalan plum hybrids Krymsk 1 and et al., 2013). Noninoculated controls were propagated as described earlier. By 3 weeks Krymsk 86). inoculated with 200 mL sterile PDB. In this postinoculation, the entire layer of the plant way, there were four inoculation treatments: growth medium became colonized by Armil- Materials and Methods Sac304, Sol310, SC4, and noninoculated laria, and the first plants began to succumb to controls. infection at 1 month postinoculation (data not Plant propagation and inoculation. Our For each of the three experiments, there shown). Nonetheless, differences in mortality approach aimed at propagating rootstocks in were six replicate plants per plant genotype · between the two rootstocks were not significant vitro and inoculating the growth medium inoculation-treatment combination (8 plant until 2 months postinoculation. At this point, with Armillaria. At 2 months postinocula- genotypes · 4 inoculation treatments · 6 noninoculated Nemaguard and Marianna 2624 tion, we tallied the number of dead plants per replicate plants = 192 total plants per exper- showed symptoms of nutrient deficiency, and rootstock and then used percent mortality as iment). Percent mortality was calculated as so the assay was ended. a measure of resistance (Baumgartner et al., the proportion of inoculated plants that died All noninoculated plants were free of 2013). Stocks of the rootstocks in tissue within 2 months postinoculation out of the Armillaria, as expected, based on no recov- culture were obtained from Duarte Nursery/ total number inoculated. Infection was con- ery of the pathogen from roots plated at Dry Creek Laboratories (Hughson, CA) and firmed by recovery of the pathogen in culture 2 months postinoculation. Also, there was confirmed before the experiment to root at the time of mortality or at 2 months post- no mortality among noninoculated plants. consistently in vitro. inoculation (whichever came first). Roots Armillaria isolates were recovered in culture Provided rootstock in vitro cultures were were separated from the plant growth medium from roots of all inoculated plants, either at maintained and proliferated on a medium by submerging the root system embedded the time of mortality or at 2 months post- consisting of Quoirin and Lepoivre basal salts within the medium in sterile water and then inoculation. (Quoirin and Lepoivre, 1977), Murashige and using a forceps and scalpel to gently scrape ANOVA identified significant differences Skoog vitamins (Murashige and Skoog, pieces of medium from the roots. Four root in percent mortality between plant genotypes 1962), 3% sucrose, 4.4 mM benzyladenine, tips per plant were plated on water agar within (P < 0.0001) and Armillaria isolates (P < 0.49 mM indolebutyric acid, and 0.6% agar 2 d of plant mortality. After 10 d of incubation 0.0001). With no interaction between plant gel. The medium was adjusted to pH 5.7 before at 25 C, cultures were examined for Armil- genotype and isolate (P = 0.9), the order of autoclaving. The medium was dispensed (50 mL) laria colonies with the following characteris- genotypes starting with the most resistant into Magenta jars (400 mL) with nonvented tics: colony diameter of 2 cm, regular colony was as follows: Krymsk 86, Krymsk 1, GA7 closures (Sigma). Proliferating cultures margin, clampless hyphae embedded in the Marianna 2624, Lovell, Empyrean 1, Nem- were maintained in a growth room with a 16-h agar, sparse white aerial hyphae, absence of aguard, Bright’s 5, and Hansen 536. On a per photoperiod (47.1 mmol·m–2·s–1, vita-Lite 40 W) spores/spore-bearing structures, and the pos- isolate basis, there were subtle differences in at 24 C. sible presence of immature rhizomorphs this order of relative resistance, especially Explants were harvested for rooting from (white when embedded in the agar, black among the most susceptible genotypes vigorously proliferating cultures. Individual when above the surface). Root tips gathered (Table 1). Nonetheless, for all three isolates, explants (1.0–1.5 cm length, with apical from noninoculated plants were examined in mean comparisons identified Krymsk 86 as meristem) were removed from proliferating this same manner on the day of inoculation having the lowest levels of mortality, clumps and placed on 1/2 strength Murashige and at 2 months postinoculation, in each of the Marianna 2624 and Lovell as having inter- and Skoog medium supplemented with 2% three experiments (n = 6 plants per genotype mediate levels, and Hansen 536 as having the sucrose, 2.5 mM indolebutyric acid, and 0.7% per experiment). highest levels (Table 2; Fig. 1). There were agar gel in 20 · 150 mm culture tubes topped Statistical analyses. Analysis of variance no significant main or interaction effects of with Kimble PM caps. Cultures were then (ANOVA) was used to determine the effects experiment (P values all $0.6). Together, the placed in the dark for 14 d at 24 C. Explants of the independent experiments (i.e., 1, 2, or Krymsk genotypes had significantly lower were then returned to the lighted growth 3), plant genotype (Bright’s 5, Empyrean 1, mortality than all other rootstocks and were chamber (16-h photoperiod) where root ini- Hansen 536, Krymsk 1, Krymsk 86, Lovell, even more resistant than the resistant control, tiation began. The rooting treatment was Marianna 2624, and Nemaguard), inocula- Marianna 2624 (63% mortality; n = 54 plants, started with an excess of explants so that 15 tion treatment (SC4, Sac304, Sol310, or summed across all three isolates and all actively growing and well-rooted clones noninoculated), and their interactions on three experiments). Marianna 2624 was

HORTSCIENCE VOL. 53(1) JANUARY 2018 5 Table 1. Parentage of clonally propagated Prunus genotypes inoculated with Armillaria. Species of plum rootstock, averaged across isolates and ex- are in boldface text. periments), is consistent with past field trials Rootstock Parentage with Prunus (Beckman et al., 1998; Beckman Bright’s 5 Peach · Almond and Pusey, 2001; Guillaumin et al., 1989). In Empyrean 1 (aka Barrier 1) Peach · Prunus davidiana one field trial, over 100 peach and plum lines, Hansen 536 [Okin. · (P. davidiana · Pe PI 6582)] · Almond planted in field soil where A. tabescens Krymsk 1 (aka VVA 1) Prunus tomentosa · Prunus cerasifera inoculum was present, were evaluated within Krymsk 86 (aka Kuban 86) Peach · P. cerasifera nine years of planting (Beckman et al., 1998). Lovell Peach The authors note that common rootstocks for Marianna 2624 P. cerasifera · Prunus munsoniana Nemaguard Peach · P. davidiana peach in the southeastern United States, Lovell and Nemaguard (propagated on a com- mercial basis as seedlings), had similar levels Table 2. Percent mortality of each plant genotype at 2 months postinoculation, for each of one isolate of of survival at 36% and 33%, respectively. We Armillaria tabescens (SC4) and two isolates of Armillaria mellea (Sac304 and Sol310) versus had comparable findings after 2 months for noninoculated controls. Each cell is the mean of 18 observations, averaged across three replicate both A. tabescens and A. mellea in our growth experiments (n = 6 plants per isolate · plant genotype, per experiment). chamber experiment, albeit for clonally prop- Percent mortality at 2 months postinoculationz agated selections of Lovell and Nemaguard, Plant genotype SC4 Sac304 Sol310 Noninoculated under much greater inoculum pressure. Krymsk 86 8.34 a 38.64 a 35.00 a 0 Armillaria mellea and A. tabescens have Krymsk 1 25.00 ab 45.00 ab 37.50 ab 0 different geographic ranges (Coetzee et al., Marianna 2624 44.22 ab 75.56 ab 69.63 ab 0 2011). As such, no field trial has evaluated Lovell 46.67 ab 85.36 ab 83.34 ab 0 these two species. Nonetheless, we can make Empyrean 1 50.00 ab 100.00 b 83.33 ab 0 some general comparisons among different Nemaguard 68.55 b 92.80 b 92.27 b 0 field trials, which share in common some of Bright’s 5 66.67 b 100.00 b 95.00 b 0 the same Prunus rootstocks. Field trials in the Hansen 536 72.92 b 94.45 b 100.00 b 0 z southeastern United States have identified Means in the same column with different letters are significantly different, based on lack of overlap of new rootstocks for peach, plum hybrids their 95% confidence intervals (P < 0.05). ‘MP-29’ (Prunus umbellata · peach) and ‘Sharpe’ (Prunus angustifolia · unknown plum species), with resistance to A. tabescens There were similar levels of mortality (Beckman, 2008; Beckman et al., 2012). In among plants inoculated with the two isolates general, their results for these two new of A. mellea, averaging 79% for Sac304 and rootstocks are in agreement with an earlier 75% for Sol310 (n = 144 plants, summed field trial in A. tabescens-infected soil in that across all eight plant genotypes and all three Prunus with various plum parents (e.g., ‘Blue experiments). Armillaria tabescens isolate Goose’ and ‘Edible Sloe’) were more re- SC4 was significantly less virulent, with 48% sistant than those with a peach background mortality (n = 144 plants, summed across all (e.g., Nemaguard) (Beckman et al., 1998). In eight plant genotypes and all three experi- our growth chamber assay, we found similar ments). All Empyrean 1 and Bright’s 5 plants results on plum parentage for both A. mellea inoculated with Sac304 and all Hansen 536 and A. tabescens: lower mortality for all three plants inoculated with Sol310 died by 2 rootstocks with P. cerasifera (aka Myrobalan months postinoculation, in all three experi- plum) as a parent (Krymsk 86, Krymsk 1, and ments (Table 2). By contrast, the highest Marianna 2624). These findings are consis- level of mortality for SC4 inoculations was tent with past research on A. mellea, in which observed in Hansen 536 at 73% (n =18 Myrobalan parentage in Marianna 2624, also plants, summed across all three experiments). shared with ‘Myran’ and ‘Ishtara’, was asso- Nonetheless, we found similar relative re- ciated with very low levels of mortality in sistance among plant genotypes for A. tabes- a field trial with controlled inoculations of cens isolate SC4 in terms of much lower A. mellea (Guillaumin et al., 1989). mortality for the Krymsk genotypes com- Our findings are in contrast to past re- pared with all others. search on A. tabescens, in which Marianna 2624, ‘Myran’, and ‘Ishtara’ all performed Fig. 1. Mortality among almond rootstocks in our Discussion worse than Lovell in a field trial with soil growth chamber assay was as follows: (A) naturally infected with A. tabescens (Beck- lowest for Krymsk 86, (B) moderate for Marianna 2624 and (C) Lovell, and (D) highest California almond growers are not satis- man and Pusey, 2001). Nonetheless, for Hansen 536. Images are representative of fied replanting all Armillaria-infected sites Marianna 2624 may have performed poorly a subset of these four rootstocks inoculated with Marianna 2624 because it is incompat- against A. tabescens in the field because of with Armillaria mellea isolate Sol310 at ible with some popular almond cultivars, interactions with abiotic factors (climate, soil 2 months postinoculation, during the first of namely, Nonpareil (Kester and Grasselly, pH, etc.); other diseases, namely, Peach tree three replicate experiments. The pathogen is 1987), the most widely planted almond cul- short life (PTSL) (Beckman et al., 1998), visible on the surface of the tissue culture tivar. It also sprouts multiple shoots (suckers) a disease complex incited by the bacterial medium as white to tan-colored aerial myce- from its roots, making harvest off the orchard canker pathogen Pseudomonas syringae lium. floor more difficult. In our evaluation of eight pathovar syringae van Hall and exacerbated almond rootstocks, Krymsk 86 had the lowest by ring nematode Mesocriconema xenoplax statistically significantly more resistant than mortality, and this finding was consistent for (Ritchie and Zehr, 1995); or both. Indeed, the susceptible control Nemaguard, which two isolates of A. mellea and one isolate of trees grafted to Marianna 2624 and other had 85% mortality. Hansen 536 and Bright’s A. tabescens, in three replicate experiments. plum rootstocks are more susceptible to 5, with 89% and 87% mortality, respec- The broad range of resistance we found bacterial canker than those on peach root- tively, were not significantly different from among the eight rootstocks, ranging from stocks (Teviotdale and Kirkpatrick, 2002), Nemaguard. 27% to 89% mortality (n = 54 plants per and Marianna 2624 has been shown to be

6 HORTSCIENCE VOL. 53(1) JANUARY 2018 very susceptible to M. xenoplax (McKenry or lower levels of mortality would, thus, be Beckman, T.G. 2008. ‘Sharpe’, a clonal plum et al., 2007). Alternatively, the A. tabescens considered resistant. rootstock for peach. HortScience 43:2236–2337. isolate we used, SC4, is highly virulent The rootstocks selected for this study Beckman, T.G., J.X. Chaparro, and W.B. Sherman. compared with the population of A. tabescens were focused on almond production, which 2012. ‘MP-29’, a clonal interspecific hybrid root- isolates present in the field trials cited earlier. is widespread in California. Certainly, some stock for peach. HortScience 47:128–131. Beckman, T.G., W.R. Okie, A.P. Nyczepir, P.L. Certainly, we selected SC4 strictly based on of the same rootstocks have been studied in Pusey, and C.C. Reilly. 1998. Relative suscep- its similar in vitro growth to our fastest the southeastern United States, namely, those tibility of peach and plum germplasm to Armil- growing A. mellea isolates from Prunus. to which peach scions are typically grafted laria root rot. HortScience 33:1062–1065. Our growth chamber assay is much more (e.g., Lovell and Nemaguard) (Beckman and Beckman, T.G. and P.L. Pusey. 2001. Field testing simplistic than a field trial and, thus, our Pusey, 2001). Our inclusion of such root- peach rootstocks for resistance to Armillaria findings are too preliminary for making plant- stocks and A. tabescens in the experimental root rot. HortScience 36:101–103. ing decisions in almond orchards just yet. design was meant to determine whether our Bliss, D.E. 1951. The destruction of Armillaria mellea Being able to complete the assay in the course growth chamber assay reflected findings from in citrus soils. Phytopathology 41:665–683. Box, G.E.P., W.G. Hunter, and J.S. Hunter. 1978. of 1 year is an advance that allows researchers previous field trials (Beckman et al., 1998). As such, it is difficult to extend our findings Statistics for experimenters: An introduction to to screen through new almond rootstocks, design, data analysis, and model building. albeit those that root well in tissue growth to peach production in the southeastern Wiley, New York, NY. medium, to select promising genotypes for United States. First, our study did not include Coetzee, M.P.A., P. Bloomer, M.J. Wingfield, validation of resistance in field trials. How- ‘Guardian’, one of the most widely planted and B.D. Wingfield. 2011. Paleogene radia- ever, replicated field trials are much better Prunus rootstocks in the southeastern United tion of a plant pathogenic mushroom. PLoS than our growth chamber assay at evaluating States (Beckman and Pusey, 2001). PTSL One 6:e28545. rootstocks in variable soils and climates, under resistance in ‘Guardian’ drives its popularity Elias-Roman, R.D., R.A. Guzman-Plazola, N.B. Klop- commercial orchard management practices, as a peach rootstock in the southeastern US, fenstein, D. Alvarado-Rosales, G. Calderon-Zavala, but it is rarely planted in California, where J.A. Mora-Aguilera, M.S. Kim, and R. Garcia- and they allow for exposure to other biotic Espinosa. 2013. Incidence and phylogenetic factors, all of which help determine the nonetheless the pathogens associated with PTSL (P. syringae pathovar syringae and analyses of Armillaria spp. associated with suitability of a rootstock. Our growth chamber root disease in peach orchards in the State of assay was reliable, in terms of infecting all M. xenoplax) cause similar symptoms Mexico, Mexico. For. Pathol. 43:390–401. inoculated plants, and brought about repeat- (Ritchie and Zehr, 1995). Second, some of Guillaumin, J.J., J. Pierson, and C. 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