451 For. Snow Landsc. Res. 76, 3: 451–454 (2001)
Evaluation of resistance of Castanea sp. clones to Phytophthora sp. using excised chestnut shoots
Josefa Fernández-López1, Rosa A. Vazquez-Ruiz-de-Ocenda2, Raquel Díaz-Vázquez1, Santiago Pereira-Lorenzo2
1 Departamento de Producción Forestal, CIFA Lourizán, Apt. 127, 36080 Pontevedra Spain
2 EPS Universidad de Santiago de Compostela, Lugo Spain fina@inia.es
Abstract
Excised chestnut shoots were inoculated with Phytophthorasp. strains to assess the resistance of 31 different Castaneaclones, including Euro-Asiatic hybrids, to this fungus. The length of the necrosis caused by the fungus is considered negatively proportional to the resistance to Phy to - phthorasp.
Clone classifications made with two different Phytophthorasp. strains were identical for the most resistant clones. Clonal heritability of resistance to Phytophthorasp. calculated for the more vigorous fungal strain was high, with a value of 0.82 for the joint analysis of the four repetitions.
Therefore the test can be replicated quite well.
Keywords: Castanea,indirect selection, genetic correlation, resistance test, Phytophthora,clonal heritability.
1 Introduction
Excised shoots of chestnut (Castanea sp.) (SALESSES et al. 1993) were inoculated with Phytophthorasp. strains to assess the resistance of different chestnut clones to this fungus.
The chestnuts included Euro-Asiatic Castaneahybrids currently under evaluation for wood production in Atlantic areas severely damaged by Phytophthorasp. The length of the necro- sis caused by inoculating excised shoots of chestnut clones with the fungus is considered to be an indirect measure of resistance to Phytophthorasp. (SALESSESet al.1993), assuming that this length is negatively proportional to the resistance to the fungus. Selection by resist- ance to Phytophthorasp. based on inoculations in excised shoots could be interesting if the heritability of this character is high and if correlations between this measure and data from evaluations of living plants are high. A test with excised shoots separates the environmental and genetic causes of variability well and can be performed at a very low cost. Replicates are easily carried out and homogeneous environmental conditions are maintained more easily than with tests in living plants.
When several clones are tested under different treatments, the clonal heritability of a trait can be estimated to determine the degree of genetic control of a character (FRAMPTON
and FOSTER 1993). High values of heritability of a trait, as estimated by several Phy to - phthoratreatments or by different repetitions, indicate whether clone classifications made for that trait are replicable.
The objectives of the experiment described here were: 1) to compare the clone classifi - cation made with two different Phytophthorasp. isolates, and 2) to determine the degree of genetic control of resistance to Phytophthorasp.
2 Materials and methods
The experiment had a factorial design with the Castaneaclone and the Phytophthorasp. treat- ments as the main factors and five replicates. The experiment was repeated four times: in June 1996, 1997 and 1998 when the shoots were still growing, and once again in September 1997, when
452 Josefa Fernández-López et al.
the shoots were completely lignified. Cuttings (30 cm long) were taken from the current year’s shoots on stumps included in the nuclear stock of hybrid clones at CIFA Lourizán. Thirty-one clones were tested. The genotypes of these clones, as determined by isozymes (FERNANDEZet al.
1995), were 13 F1C. crenatax C. sativacrosses, 1 F1of C. mollisimax C. sativa,eight offsprings of a C. crenatax C. sativacross with unequal proportions of each parental species, presumably F2
hybrids, and one clone, CHR-11, with isozyme bands of C. crenata,although it is considered a second or third generation hybrid. Eight pure species were included as controls: three C. sativa clones (sensitive controls), three C. crenataand two C. mollissima.It was hypothesized that the latter five clones were resistant to Phytophthorainfection.
The Phytophthora treatments had three levels. The plants were inoculated with: i) T1, a Phytophthora sp. isolated from the heavily infected nursery soil of Lourizán, with a similar morphology to that of P. cinnamomiRands, ii) T2, P. cinnamomiCBS341.72 (A2) and iii) T3the pea-agar medium (DINGRAand SINCLAIR1985) as a control. The two Phytophthorasp. strains were cultivated on pea-agar medium. Inoculations were made by inserting an agar plug (Ø 5 mm) taken from the margin of an eight-day-old culture into an incision applied to the upper part of the cutting. The incision was covered with an aluminium sheet to avoid desiccation. Inoculated cut- tings were inserted in trays with perlite and kept in a tunnel inside a greenhouse at temperatures between 20 and 25 ºC and a relative humidity close to saturation. Resistance to Phytophthorasp.
was evaluated by measuring necrosis length (L) in centimetres at 14 and 21 days after inoculation.
The data from each repetition were analysed with PROC GLM in SAS (1989) using the model Lijk= μ+ Ci+ Tj+ CTij+ εk(ij), where Ciis the effect of the clone factor (i = 1–31); Tjis the effect of the treatment with Phytophthorasp. (j = 1–3); CTijis the effect of the interaction between clone and treatment; and εk(ij)is the error term, which, in this case, is the variation within a clone. In this first analysis all the factors were considered fixed with the objective to find out if there were sig - nificant differences between treatments T1and T2and the control T3. A second ANOVA was made to estimate variance components with the RANDOM option of GLM procedure in SAS (1989) using the precedent model. Data from the controls, T3, were excluded from this analysis because the objective was simply to estimate variances caused by T1and T2. In this case the clone factor and the interaction clone by treatment were considered to be random and the treatment fixed.
For estimating of the clone heritability, the joint analysis of the four repeats was made separately for each Phytophthorasp. treatment. The model for the analysis was Lijk= μ+ Ci+ Dj+ CDij+ εk(ij), where Djis the effect of date of repetition of the experiment (j = 1–4) and CDijis the effect of the interaction between clone and date of each repetition. Clonal heritability was calcu- lated as a broad sense heritability or heritability of clonal means (FRAMPTONand FOSTER1993) as H2C= k2σ2C/ [k2σ2C+ k1σ2CD+ σe2], where σ2Cis the variance among clones, σ2CDthe variance due to clone-date interaction, σe2the variance due to error, and k1and k2the coefficients of vari- ances σ2CDand σ2Cin the expected mean squares of the ANOVA.
Type B genetic correlation was calculated using the method given in YAMADA (1962) to compare the evaluations made in September with lignified shoots and the evaluations performed in June, when the shoots were still growing.
Clone classifications by resistance to Phytophthorasp. were established separately for each strain, T1and T2, by the multiple test means Student-Newman-Keuls. Original data were stan- darized before performing the means test according to Li- μ/ σ. μand σare the mean and the standard deviation for each repetition of the experiment.
3 Results and Discussion
The length of necrosis caused by inoculation with Phytophthorasp. is significantly affected by all factors of the model (clone, treatment and the interaction of both). The most im - portant factor is the treatment. Mean values of necrosis length of the Phytophthora sp.
treatments were always higher for the local strain T1than for T2, but statistically different from the control T3(Fig. 1). The differences between T1and T2steadily increased from 1996 to 1998.
453 For. Snow Landsc. Res. 76, 3 (2001)
In 1996 clone classification based on lesion length was exactly the same for the measure- ments after 14 and 21 days and was also very similar in June 1997, with only insignificant changes. Evaluation after 21 days showed a higher genetic component (σ2C) than after 14 days in 1996, but in June 1997 the opposite results were obtained (Table 1).
It can be concluded that the duration of the test, measured in days after inoculation, was not very critical. In sensitive clones the length of necrosis was close to 20 cm in juvenile shoots and between 13 and 18 cm in lignified ones, while in resistant clones it was always shorter than 5 cm.
Table 1. Components of variance for each repetition of the experiment. L is the necrosis length, sub- indexes indicate the month in which the experiment was performed (J for June or S for September), the number of days between inoculation and measurement (14 or 21 days) and the year of the experiment (96, 97, 98). σ2Cis variance among clones. In brackets is the percentage of variance associated with each factor in relation to the phenotypic variance. σ2CTis variance due to the interaction clone-treatment, σ2e
is the error variance and σ2Pis the phenotypic variance calculated as σ2P= σ2C+ σ2CT+ σ2e. Significance level of the F statistic are represented by either ns (non significant) or *, **, *** for significant at P< 0.05, 0.01, 0.001, respectively.
Variable σ2C σ2CT σ2e σ2P
Lj14–96 33.66 (45.9)** 4.63 (6.3)*** 34.99 (47.7) 73.3
Lj21–96 52.3 (52.8)*** 5.17 (5.2)*** 41.53 (41.9) 99.0
Lj14–97 4.73 (16.7)* 4.14 (14.6)** 19.38 (68.6) 28.2
Lj21–97 11.62 (11.1)* 22.77 (21.7)** 70.58 (67.2) 104.9
Ls14–97 6.42 (15.5)* 7.22 (16.6)*** 25.14 (64.8) 38.8
Lj14–98 1.82 (6.6)ns 9.28 (33.3)*** 16.71 (60.1) 27.8
Estimation of the variance components for each repetition showed that the genetic com- ponent σ2Cdiminished from 1996 to 1998. This is explained mainly by the increasing inter - action σ2CT(Table 1) caused probably by the reduced vigour of T2(Fig. 1).
The clonal heritability value (0.82) is very high for evaluations with the local PhytophthoraT1, but much lower for T2(0.41) (Table 2). Therefore, the degree of genetic control of the length of necrosis caused by the local Phytophthorasp. is very high.
The fact that the clone-treatment interactions were significant, means that clone classifi- cations made with the two Phytophthorasp. strains differ to a certain extent. However, the comparison between clone classifications made with data from both strains are completely in agreement in the group of clones classified as more resistant. This group consists of the interspecific hybrids CHR-33, CHR-35, CHR-93, CA-15, CHR-177, CHR-27, CHR-162, Fig. 1. Resistance test with Phytophthora sp.
Castanea sp. cuttings from 31 sensitive and resistant clones were inoculated with T1 = Phytophthorasp., T2 = P. cinnamomi,T3 = con- trol. Mean values and standard deviations of necrosis length 14 and 21 days after in oculation are given. See Table 1 for abbreviations.
Year and month of treatment 35
30 25 20 15 10 5
0 Lj 14 –96 Lj 21–96 Lj 14 –97
L (cm)
Lj 21–97 Lj 14 –97 Lj 14 –98 T1 T2 T3
454 Josefa Fernández-López et al.
CHR-32 and the two C. crenata,B-32 and B-33. The C. mollissimaclones ID4L, D-3 and the C. crenata clone B-9 changed their position in the classifications made with T1 and T2, although not radically.
Table 2. Clonal heritability (H2C= k2 σ2C/ [k2σ2C+ k1σ2CD+ σe2]) of evaluations performed with treatments T1and T2calculated for the joint analysis of four repetitions. k1= 4.9 and k2= 15.8. For ab - breviations see Table 1. In brackets the percentage of variance associated with each factor in relation to the phenotypic variance. σ2CDis variance due to clone-date interaction. The phenotypic variance is σ2P= σ2C+ σ2CD+ σ2e.
Variable σ2C σ2CD σ2e H2C
T1 27.27 (32.5) *** 10.35 (12.3) *** 46.32 (55.2) 0.82
T2 3.68 (8.2) * 10.8 (24.1) *** 30.41 (67.7) 0.41
Bigger changes occurred in the extremely sensitive clones. The C. sativaclones 25, 41 and 6153 were classified as extremely sensitive with the local strain T1, but only clone 41 was very sensitive in the classification made with T2. The clones classified as rather sensitive with both strains were the hybrids CHR-167, CHR-168, CA-07, CHR-98, CHR-14 and the clone CHR-11. All other clones were in intermediate positions in both classifications.
Genetic correlations between the evaluations in September 1997 and the evaluations in June 1996, 1997 and 1998 were high with values of 0.72, 0.79 and 0.85. Therefore, evaluations made when shoots were completely lignified were similar to those performed when shoots were still growing.
Inoculation of cuttings seems to be a good method to test resistance to Phytophthorasp.
However, the method needs to be compared with direct measurements obtained by inocu- lating the roots of living plants or with plant survival in field trials in heavily infected soils.
With clones planted in areas infected by Phytophthorathere was a correspondence between survival and the presented data, especially for the C. sativaclones (unpublished results).
Several conclusions may be drawn from these findings: i) clone classifications made with two different Phytophthora sp. strains were identical for the most resistant clones, ii) Castaneahybrids were classified from sensitive to resistant, while C. sativaclones were sensi- tive to slightly sensitive and the Asiatic species were resistant to slightly resistant, and iii) the length of the necrosis caused by inoculating of excised stems with Phytophthora remaind fairly constant.
Acknowledgements
This study was carried out within the Spanish National project FO96-031 on “Characterization of chestnut forest reproduction materials for use in reforestation”.
5 References
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Accepted 2.4.02
