Evaluation of Chestnut Susceptibility to Cryphonectria parasitica: Screening under Controlled Conditions

Abstract

Cryphonectria parasitica (Murrill) M.E. Barr (Sordariomycetes, Valsaceae) is the causal agent of chestnut blight. This disease is a major concern for chestnut cultivation in Europe. The fungus colonizes vascular tissues and evolves generating cankers causing severe dieback and the death of the tree. Excised and debarked well-lignified shoots of 28 C. sativa genotypes (assay A) and of 10 progenies (assay B) were inoculated with C. parasitica strain FMT3bc2 (vcg: EU2). Fungal growth was measured along the longitudinal axis on the 3rd and 6th days after inoculation. Results indicated the inoculation methodology works and the results were clear after 6 days. Differences in susceptibility to chestnut blight among C. sativa trees of Montseny have been detected both at the individual genotype level and at the progeny level. Nineteen genotypes and four progenies showed a susceptibility to Blight not significantly different from C. mollissima. The methodology was easy to apply in extensive/preliminary selection screenings to assess the susceptibility of C. sativa materials to the Blight.

1. Introduction

The Ascomycete fungus Cryphonectria parasitica (Murrill) M.E. Barr (Sordariomycetes, Valsaceae) is the causal agent of chestnut blight, one of the most devastating diseases affecting Castanea sp. worldwide. The disease in Europe was first detected in Italy in 1938 (EPPO current status: Present) [1,2] from where it spread to the neighboring countries of Switzerland (1948; widespread), Slovenia (1950; restricted distribution), and France (1956; restricted distribution) [3,4]. The disease is currently present in Spain (1947; restricted distribution), Croatia (1955; widespread), Albania (1967; widespread), Serbia (1975; widespread), Portugal (1989; widespread) and Germany (1992; restricted distribution), Azerbaijan (2004; present), United Kingdom (2011; restricted distribution), and Belgium (2014; restricted distribution) [1,5]. Most of the European C. parasitica populations seem to have originated from single introductions since they have reduced genetic diversity [6].

Spore dispersal by air is a major dissemination mechanism of fungal pathogens [7,8]. In the case of C. parasitica, air-borne spores usually penetrate above-ground parts of the host through bark wounds (e.g., pruning, wind, hail, etc.) in shoots or stems. The fungus colonizes vascular tissues causing progressive wilting, and severe reduction in fruit production. Later symptoms include orangish necrotic areas in the bark [2,9]. The necrosis evolves generating cankers that usually girdle branches or even the trunk causing severe dieback, and finally the death of the tree [10,11]. Cankers harbor fruiting bodies of the fungus that disseminate their spores throughout the chestnut stands. The fungus infects Castanea sativa Mill. (Europe), Castanea dentata (Marshall) Borkh. (America), as well as Castanea crenata Siebold and Zucc. and Castanea mollissima Blume (Asia) at any age. Among these host species, C. sativa and C. dentata are considered the most susceptible since C. mollissima and C. crenata exhibit some tolerance to the disease probably caused by ancient co-evolution with the pathogen in their native range [12,13].

This disease is a major concern for chestnut cultivation in Europe as there is currently no effective cultural or chemical control and no resistance has been found in C. sativa species [14]. To find a way out of chestnut blight, two main strategies are being considered: biocontrol through virus-induced fungus hypovirulence, and prevention by breeding disease-resistant genotypes. The use of RNA mycoviruses (i.e., Cryphonectria hypovirus 1; Hypoviridae) has resulted in highly effective in Europe [1], thus becoming one of the most successful alternatives for controlling the disease in natural stands. This biocontrol method is based on inoculation of virus-infected fungal strains belonging to the same vegetative compatibility group/s (vcg) than those causing disease in the target stand [15,16]. This infected—and therefore weakened—strain, inoculated in diseased trees, naturally transfers viruses by mycelial anastomosis to virulent strains which will become in turn hypovirulent. Mycovirus-infected strains of C. parasitica are not able to severely damage the tree allowing it to withstand the infection with minor symptoms [17,18]. On the other hand, breeding resistant genotypes of chestnut for planting is probably the most promising preventive measure to be implemented for woody croplands and afforestation. The transfer of resistance from Asian chestnuts to American or European species has been carried out in several programs but reaching the desired fruit quality requires several backcrosses that lengthen the selection process considerably [19,20]. Finding resistance or lower susceptibility traits in natural populations of C. dentata, the American chestnut, has proven possible [21] although conducting this work by inoculation of standing trees requires a great deal of work that is often not feasible. The evaluation of tolerance/resistance to biotic or abiotic injuries under controlled conditions on parts of plants/trees has proven to be an advantageous practice when many individuals/samples are to be tested. Thus, in different hardwoods similar approaches have been used, such as on Juglans sp. to study frost resistance [22] or more specifically on chestnut to establish levels of susceptibility to Phytophthora spp. [23]. Moreover, Rodríguez and Colinas [24] showed that estimating chestnut susceptibility to C. parasitica by means of inoculation on excised shoots was feasible and the mycelium grew as expected under controlled conditions.

In consequence, the aims of this study were: (i) to evaluate the tolerance to chestnut blight of some C. sativa genotypes from a Spanish local population (Montseny-Northeast of Catalonia) in excised shoots, and (ii) to study this tolerance in seedling genotypes of the same origin installed in a progeny test.

2. Materials and Methods

2.1. In Vitro Inoculation: Budstick Assay

2.1.1. Assay A

Tolerance to chestnut blight among C. sativa genotypes was evaluated in vitro by inoculating a virulent strain of C. parasitica in debarked well lignified shoots. One-year-old budsticks of 28 C. sativa genotypes (

Table 1. Description of genotypes used in the in vitro assays (A and B). G: mean daily growth of the Cryphonectria parasitica colony from inoculation to the sixth day of incubation along the longitudinal axis. Data for genotypes of Castanea mollissima in assay A and B are shown in the left and right sides of the bar (/) respectively. n: number of replicates per genotype. Mean values and standard deviation are shown.

Species	Assay	Genotype/Progeny	N Genotypes	n	Cutting Diameter (cm)	G (mm/Day)
Castanea mollissima	A/B	M2	1	8/4	0.84 ± 0.06/0.79 ± 0.14	3.9 ± 1.2/5.8 ± 2.5
		M5M	1	8/4	1.14 ± 0.05/0.60 ± 0.06	4.6 ± 1.4/4.6 ± 2.7
Castanea sativa	A	BRL02	1	8	1.41 ± 0.08	5.6 ± 1.2
	A	BRL03	1	8	0.90 ± 0.03	4.4 ± 1.7
	A	BRL06	1	8	0.98 ± 0.13	4.9 ± 1.4
	A	CNV01	1	8	0.91 ± 0.07	5.8 ± 1.2
	A	FGM01	1	8	1.21 ± 0.08	5.4 ± 1.5
	A	FGM04	1	8	1.43 ± 0.15	5.4 ± 1.8
	A	FGM11	1	8	0.93 ± 0.10	6 ± 1.2
	A	MSY03	1	8	0.95 ± 0.06	5.8 ± 1.7
	A	MSY04	1	8	0.89 ± 0.09	5.4 ± 0.9
	A	MSY08	1	8	0.91 ± 0.04	5.6 ± 2.2
	A	MSY09	1	8	0.93 ± 0.08	5.3 ± 1.8
	A	MSY10	1	8	1.06 ± 0.08	3.9 ± 1.6
	A	MSY11	1	8	1.08 ± 0.04	5.5 ± 0.7
	A	MSY12	1	8	1.16 ± 0.38	5.9 ± 0.8
	A	MSY14	1	8	1.24 ± 0.12	4.3 ± 2.0
	A	PO11	1	8	1.13 ± 0.13	4.5 ± 1.3
	A	RLL05	1	8	1.03 ± 0.12	4.5 ± 1.1
	A	RLL10	1	8	0.96 ± 0.20	6.4 ± 1.1
	A	SPV05	1	8	1.35 ± 0.06	5.9 ± 1.8
	A	VLD12	1	8	1.01 ± 0.08	5.7 ± 1.1
	A	VLD14	1	8	1.23 ± 0.10	5.4 ± 1.3
	A	VLD22	1	8	1.14 ± 0.05	5 ± 0.9
	A	VLD29	1	8	1.11 ± 0.04	5.4 ± 1.0
	A	VLD30	1	8	1.19 ± 0.10	4.4 ± 1.7
	A	VLD31	1	8	1.00 ± 0.11	6 ± 1.5
	A	VLD32	1	8	1.02 ± 0.04	5.3 ± 1.6
	A	VLD33	1	8	1.11 ± 0.06	6.1 ± 1.3
	A	VLD34	1	8	1.21 ± 0.11	5.5 ± 1.9
	B	CS-49	23	4	0.71 ± 0.13	6.7 ± 1.7
	B	CS-51	14	4	0.69 ± 0.11	6.9 ± 1.8
	B	CS-52	12	4	0.69 ± 0.11	8 ± 1.4
	B	CS-54	17	4	0.71 ± 0.09	6.9 ± 1.5
	B	CS-55	24	4	0.69 ± 0.11	7.7 ± 2.1
	B	CS-56	14	4	0.69 ± 0.12	8.1 ± 2
	B	CS-57	5	4	0.72 ± 0.14	8.8 ± 1.3
	B	CS-60	20	4	0.68 ± 0.11	7.1 ± 2.1
	B	CS-61	18	4	0.66 ± 0.12	7.2 ± 1.8
	B	CS-62	18	4	0.69 ± 0.09	6.9 ± 2

) were removed from healthy mother plants installed in a quarantine greenhouse of Gimenells (Lleida, 41°44′45.6″ N; 0°23′39.1″ E) at IRTA experimental station in July 2020. The harvest date was adjusted to the optimal physiological state of chestnut for the susceptibility test [24]. Budsticks (12–15 cm long) were kept at 4 °C covering the apical ends with Parafilm^© while the opposite side was submerged in distilled water until processed. Moreover, lignified shoots from two genotypes of C. mollissima were collected at Lourizan Forest Center of Galicia (42°24′35″ N—8°00′12″ W) and sent overnight to be used as a tolerant control (Table 1).

Inoculation was performed according to the methodology of Rodríguez and Colinas [24] with minor modifications. Each budstick was cut in 7 cm sections (8 replicates per genotype; 224 samples of C. sativa and 16 samples of C. mollissima) (Figure 1) in the laboratory with disinfected pruning scissors. The diameter of each cutting sample (d) was measured with a digital caliper and the section manually debarked using a sterile scalpel. Fungal cultures of C. parasitica strain FMT3bc2 (vcg: EU2) grown at room temperature for 5 days in PDA medium (potato-dextrose-agar 3.90% w/v; Biokar) were used as an inoculum source. More specifically, a 5 × 5 mm plug of a solid medium plug with fresh mycelium from the edge of the colony was placed in the center of each debarked shoot section with the mycelium side in touch with wood tissue (Figure 1).

Inoculated samples were labeled with white tape and randomly placed in disinfected plastic trays lined with filter paper (Figure 1). Each tray was then moistened by spraying deionized water and covered with a plastic film to maintain high relative air humidity inside the tray. The trays were protected with a sheet of paper and incubated at room temperature (24 ± 2 °C) for one week. Whenever the filter paper became dry, deionized water was sprayed in the trays. Fungal growth was measured along the longitudinal axis (mm) on the 3^rd and 6^th days after inoculation.

2.1.2. Assay B

In parallel to assay A, a total of 166 C. sativa genotypes belonging to 10 progenies (Table 1) were used for an additional in vitro study. Tree shoots (4 replicates per seedling) were collected in a progenies test orchard located in the Parc Natural i Reserva de la Biosfera del Montseny (41°45′46,6″ N; 2°25′39,1″ E) and were processed and incubated as previously described for bioassay A. The same two C. mollissima genotypes, four shoots of each one, were also used as control samples. The fungal growth rate was calculated as previously explained.

2.2. Statistical Analysis

Data obtained after six days of incubation were analyzed for both in vitro assays using the programming environment R [25]. We used the average value of the mean daily fungal growth rate for a six-day incubation period (G, mm/day) of both C. mollisima genotypes (i.e., “M2” and “M5M”) as reference for model computation (control). This was tested by fitting two preliminary generalized linear models (GLMs) (one per dataset) in order to evaluate whether G significantly varied between both C. mollissima genotypes.

In assay A, a GLM was fitted as a null model including the mean daily fungal growth rate (G) as a response variable and the genotypes as an explanatory factor. Then, a linear mixed-effects model (LMM) was computed using the “lme4” package in R [26] including G as the dependent variable, genotype as a fixed factor and the diameter of the cutting (d) as a random factor (

Table 2. Results of models (GLMs and LMMs) describing the variation of mean daily lesion growth (G; mm/day) with progeny, seedling-genotype, and diameter (d; mm) in the in vitro assay. Random factors of LMMs are shown in brackets. n: number of observations used for model fitting. Selected model in bold.

Assay	Model	n	Description	Df	logLik	Deviance	AIC	ΔAIC
A	Ma0	232	G~genotype	30	−403.73	807.46	867.46	0
	Ma1		G~genotype + (d)	31	−403.73	807.46	869.46	2
B	Mbp0	664	G~progeny	12	−1355	2710	2734	0
	Mbp1		G~progeny + (d)	13	−1351.80	2703.70	2729.70	−4.30
	Mbg0		G~seedling-genotype	167	−1161.40	2322.70	2656.70	0
	Mbg1		G~seedling-genotype + (d)	168	−1161.40	2322.70	2658.70	2

). Both models were compared using Akaike’s Information Criteria (AIC) [27] using the “AICcmodavg” package in R [28]. The most parsimonious model was selected using AIC and the result of the model comparison using the χ² test.

In assay B two different model sets were fitted (Table 2). A null GLM was computed with G as a response variable and progeny as an explanatory factor. Then, an LMM with the same variables as well as diameter as a random factor was computed (Table 2). Analogous models (i.e., null and LMM) were fitted for the same dataset including the seedling genotypes instead of progenies as explanatory variables. Definitive models were selected following the same statistical indicators as described for assay A.

3. Results

The generalized linear model fitted for assay A showed significant variation in G among the evaluated materials (p-value = 0.03) (model Ma0). The LMM fitted to dataset A including diameter as a random factor (i.e., Ma1) retained the significance of genotype (p-value = 0.03). The most parsimonious model (Ma0) did not show conclusive differences with Ma1 according to AIC values (ΔAIC_Ma1-Ma0 = 2; Table 2), so we compared them with a χ² test (p-value = 1). Consequently, Ma1 was selected as the most explicative model for assay A (Table 2). The parameters of the model showed that in nineteen of the assayed genotypes (i.e., “BRL02”, ”BRL03”, ”BRL06”, “FGM01”, “FGM04”, “MSY04”, “MSY08”, “MSY09”, ”MSY10”, “MSY11”, “MSY14”, “PO11”, “RLL05”, “VLD14”, “VLD22”, “VDL29”, “VLD30”, “VLD32” and “VLD34”) the daily lesion growth rate (G) was not significantly different from the control C. mollissima (Figure 2).

In the dataset of assay B, two null models (GLMs) were computed to evaluate variations of G in respect of progenies (model Mbp0) and genotypes (model Mbg0). Both models showed a significant effect of the corresponding explicative factors (p-value < 0.01 in all cases). The LMM fitted to analyze the variation of G among genotypes including diameter as a random factor (model Mbg1) retained the significance already shown by the corresponding null model Mbg0. In addition, change in model AIC was not noticeable (ΔAIC_Mbg1-Mbg0 = 2) and both models did not differ significantly (p-value = 1). In contrast, the fitted LMM Mbp1 that included diameter as a random factor resulted more parsimonious than the corresponding null model (ΔAIC_Mbp1-Mbp0 = −4.3) and was significantly different from Mbp0 (p-value = 0.01). In consequence, either Mbp1 or Mbg1 were selected as the most explicative models for this in vitro assay (Table 2). Both models supported the lower susceptibility of C. mollissima against C. parasitica (Figure 3 and Figure S1).

According to this model, the tolerance to C. parasitica of progenies “CS-49”, “CS-51”, “CS-54”, and “CS-62” is not significantly different from that of tolerant C. mollissima making them good candidates for further studies regarding their suitability to be planted in orchards. The use of average G values for C. mollissima genotypes as a reference in model fitting was supported by the lack of significant differences of G between “M2” and “M5M” in any of the datasets (p-value > 0.20 in all cases).

4. Discussion

In this assay, C. parasitica colonized the wood in the first six days after inoculation in contrast with other methodologies where longer periods were required [29]. The methodology used, debarking of lignified shoots, was expected to improve colonization by the fungus compared to inoculation on live seedlings [30] or on stems with bark [13]. In addition, working under controlled conditions allows to not have to worry about the potential infection and death of the plant, or part of the plant, or introducing new vcgs potentially foreign to an area. Future research involving inoculation with fungi from different populations—even in simultaneous infection—will be informative to characterize the most resistant genotypes suitable for establishing new plantations. However, an aspect to be considered is the virulence of the fungal strain used which is expected to vary among isolates [31]. In this study, we only tested one fungal strain since our main objective was to characterize host susceptibility against a strain of the EU2 vcg, the most common vcg type in the Montseny area [32].

Regarding features of plant material, Pažitný et al. [12] reported a significant effect of stem diameter on chestnut blight lesion development. This observation completely agrees with our fitted models (Table 2) where the inclusion of stem section diameter provided a better explanation of recorded data.

Nearly all the C. sativa materials included in this study come from a reduced population of chestnut trees of the Montseny Natural Park, therefore, extending this study to a wider genetic base would be the first step to take in future evaluations. “PO11” is the only material not originating from this population, it is a spontaneous chestnut hybrid selected as a rootstock of C. sativa [33] for its resistance to Phytophthora spp. In this evaluation “PO11” has been included in the group whose tolerance is comparable to the one of C. mollissima, which was expected since this genotype carries some Asian genes [34].

It should be noted that 70% of the C. sativa genotypes and the 40% of progenies from Montseny inoculated with C. parasitica showed a response analogous to the one of C. mollissima in their canker development. These results are relevant because the chestnut orchards in Montseny are not described as having been introgressed with Asian materials as in other Spanish areas [35]. Their ancestry and traditional management in the last 100 years do not indicate the entry of foreign fruit varieties. Nor are there any genotypes with proper names that could suggest that they are ancient varieties of the Montseny area. Chestnut trees sampled from this area, according to Mattioni et al. [36], clustered (Nei distance) together with trees from southern Italy and Sicily but not with those of bordering areas. Southern Italy chestnuts have genetics from the Eastern Mediterranean [36]. Perhaps by going deeper into the origin of the Montseny materials, it would be possible to identify other materials to include in further C. parasitica susceptibility screenings.

5. Conclusions

Differences in susceptibility to chestnut blight among C. sativa trees have been detected both at the individual genotype level and at the progeny level. The debarked budstick-based inoculation method used here revealed nineteen genotypes and four progenies of C. sativa with a susceptibility to C. parasitica not different from that of C. mollissima.