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Introduction
The gall wasp Andricus quercuslaurinus Melika & Pujade-Villar has two stages in its life cycle: the sexual one that produces galls on leaves and the asexual one that causes galls on branches and generates the downward death of its main host Quercus affinis Scheidw. (Melika et al., 2009). Since December 2005, this cynipid insect is responsible for the death of at least 80 % of the natural population of Q. affinis in Acaxochitlán, Hidalgo, Mexico (Barrera-Ruiz et al., 2016). The National Forestry Commission (CONAFOR) of Mexico conducted aerial spraying using Spirotetramat insecticide in 2012 and 2015 to decrease the wasp population in the sexual generation (Barrera-Ruiz et al., 2016). From April to November 2018, 1 812.86 ha were affected by the gall wasp. As a result, a phytosanitary contingency was declared, and comprehensive control measures were established for A. quercuslaurinus (Sistema Integral de Vigilancia y Control Fitosanitario Forestal [SIVICOFF], 2018).
The extent of the impact is evaluated with descriptive keys or rating scales. These keys have scales with levels that translate to percentage and degrees of resistance and qualitative levels such as mild, moderate and high (Alfenas et al., 2009). Pujade-Villar et al. (2018) indicate that wasp control, in the long term, can be achieved with management by resistance or tolerance to attack. In this regard, Velasco-González (2019) determined that 14 % of the Q. affinis population of the ranch La Victoria, Acaxochitlán, Hidalgo, is tolerant to the pest.
Vegetative propagation helps to generate attack-resistant individuals, because they retain the genotypic characteristics of the donor tree (Hartmann et al., 2014). In addition, grafting shortens the period for seed production and establishment of seed orchards of Q. affinis resistant to gall wasp (Kita et al., 2018; Loewe-Muñoz et al., 2022). The objective of the present study was to establish the necessary conditions for grafting of Q. affinis individuals tolerant to A. quercuslaurinus attack.
Materials and Methods
Study Area
The scions were collected at the private property La Victoria, Acaxochitlán, Hidalgo, central point at coordinates 20° 10' 07.16” N and 98° 11' 54.00” W at 2 164 m elevation. Ten juvenile and mature Q. affinis ortets (donor trees) were selected: five tolerant and five susceptible to Andricus quercuslaurinus attack. The adult trees were 35 to 40 years old with averages of 30 m in height and 95 cm to 120 cm in diameter. The young trees were 12 years old with 10 to 15 cm in diameter and 8 to 10 m in height.
Incidence scale of the Andricus quercuslaurinus attack
The incidence of wasp attack was defined according to crown transparency and the appearance of epicormic buds on stem and branches. Using representative images of the attacked trees, a scale with six damage classes was established. Classes 1 and 2 were considered tolerant phenotypes and classes 3, 4, 5 and 6 susceptible phenotypes to A. quercuslaurinus attack (Table 1; Figure 1).
Table 1 Characteristics of the damage assessment scale in Quercus affinis caused by Andricus quercuslaurinus.
| Scale | Crown transparency (%) | Shoots on stem and branches |
|---|---|---|
| 1 | 0-10 | No epicormic shoots |
| 2 | 11-20 | With or without epicormic shoots |
| 3 | 21-40 | With or without epicormic shoots |
| 4 | 41-50 | With epicormic sprouts |
| 5 | 51-99 | With epicormic sprouts |
| 6 | 100 | Dead tree |
Figure 1 Qualitative scale of attack by Andricus quercuslaurinus according to crown transparency and appearance of epicormic shoots on Quercus affinis: 1) low attack; 2) moderate-low attack, appearance of epicormic shoots and crown decline; 3) moderate-medium attack, presence of epicormic shoots in stem and crown transparency; 4) moderate-high attack, epicormic shoots in crown and high transparency; 5) high attack, downward death of the crown with epicormic shoots in stem; 6) severe attack, downward death of the tree without appearance of epicormic shoots in stem.
Grafting on Quercus affinis
The experiment was carried out in a greenhouse of the nursery of the Postgraduate Forestry Sciences Department of the Colegio de Postgraduados in Texcoco, Estado de Mexico, located at 19° 27’ 38.25’’ N and 98° 54’ 23.91’’ W at an altitude of 2 240 m above sea level.
Three experiments of apical fissure (experiment 1, 4 and 5) and two of side-veneer (experiments 2 and 3) grafts were carried out (Table 2). Grafting was carried out with herbaceous, semi-woody and woody scions of tolerant and susceptible phenotypes. The number of grafts per donor tree varied because many scions were thin and small budded, i.e., lacked vigor and were also lignified. The selected scions had high vigor, semi-woody consistency, short internodes and large buds at the end of dormancy. The tolerant and susceptible trees were grafted equally. Three experiments were conducted in 2020 (summer: 8 September, 8 October and 10 November) and two in 2021 (winter [4 March] and autumn [28 September]).
Table 2 Phenotypes, rootstock and scion characteristics in Quercus affinis grafts.
| Experiment | Datum | Phenotypes | Grafts | Number of grafts | Type of scion | Type of bud |
|---|---|---|---|---|---|---|
| 1 | 2020-09-08 | 1FT | Q. affinis | 15 | Semiwoody from young trees | Large buds at the end of dormancy (light green) |
| 2FT | Q. affinis | 10 | ||||
| 1FS | Q. affinis | 15 | ||||
| 2FS | Q. affinis | 10 | ||||
| 2 | 2020-10-08 | 3FT | Q. affinis | 10 | Herbaceous with basal shoots | Small buds with long internodes (light green) |
| 3FS | Q. affinis | 10 | ||||
| 3 | 2020-11-10 | 4FT | Q. affinis | 20 | Semiwoody of adult trees | Large buds without budding (green-brown) |
| 5FT | Q. affinis | 10 | ||||
| 4FS | Q. affinis | 20 | ||||
| 4FS | Q. affinis | 10 | ||||
| 4 | 2021-03-04 | 4FT | Q. affinis | 20 | Semiwoody of adult trees | Large buds in final dormancy (dark green) |
| 4FS | Q. affinis | 20 | ||||
| 5 | 2021-09-28 | 4FT | Q. rugosa | 16 | Semiwoody of adult trees | Large buds and at the end of dormancy (light green) |
| 5FT | Q. rugosa | 16 | ||||
| 4FT | Q. affinis | 16 | ||||
| 5FT | Q. affinis | 16 |
Collection and preparation of scions
In young and adult trees, scions were collected from the top of the canopy on twigs from the last year of growth. Basal shoots were obtained from 12-year-old trees felled in February; eight months later, stumps had herbaceous and lignified shoots. Scions were placed in sealed Ziploc® bags and placed in coolers with cooling material. Prior to grafting, the scions were cut to 10 cm in length with three or four knots. The scions were washed with toothbrush (to avoid damaging the buds) and Roma® soap powder; subsequently, they were rinsed with distilled water and disinfested for 10 min in solution with Tilt® 250CE fungicide (Propiconazole) at a dose of 5 mL∙L-1 of water. Two leaves cut in half were left on each scion to reduce transpiration and dehydration. The scions were placed on paper sheets to dry under shade and stored in icebox until grafting.
Rootstock preparation
In the first four experiments, eight-month-old Q. affinis plants were used as rootstock and in the fifth experiment, Q. rugosa and Q. affinis rootstocks (Table 1). The seed used to produce Q. affinis rootstocks was obtained from the GUMAIR nursery in the region of Acaxochitlán, Hidalgo. Transplanting was done after six months in 4 L bags with a substrate based on 60 % of oak bark and 40 % of composted pine bark. At the time of grafting, the rootstocks measured 50 cm in height and 1.5 cm in average diameter. The Q. rugosa rootstocks were two years old, 50 cm in height and 2 cm in average diameter and were in 2 L black polyethylene bags with a substrate mixture of oak bark and compost in a 3:1 ratio. The plant was produced at the Finca M Y M SPR de RL nursery in Zacatlán, Puebla, located at 19° 59' 48.43" N and 97° 59' 26.56" W.
Yara® (DAP) 18-46-00 (N-P-K) slow-release fertilizer plus micronutrients (1 g∙L-1) and Trichoderma harzianum strain ISF13 (2.5 x 104 conidia∙mL-1) dissolved in water were applied to each rootstock. Subsequently, lateral branches were removed from the rootstocks and the apical shoot was left. Quaternary ammonium salts were applied as a disinfectant in the area where the cutting was performed.
Grafting technique and management
In experiments 1, 4 and 5, the terminal fissure was used and in 2 and 3, side-veneer grafting was performed with Victorinox® knife (Table 2). The wound was secured with Parafilm®. The graft was covered with a plastic bag saturated with humidity, tied at the bottom to maintain the microclimate. Irrigations were made every four days and T. harzianum strain ISF13 was applied at a concentration 2.5 x 104 conidia∙mL-1 at the time of grafting and 20 days later. Fertigation was done with YaraMila COMPLEX and DAP (5 g∙L-1 both), alternating every 15 days in the first month after grafting and every 30 days for the next three months. Shoots that emerged from the rootstock were cut to reduce competition for water and nutrients with the grafted scion. To avoid dehydration of the graft buds, the microclimate was removed progressively: first one corner above the plastic bag was cut off, on the fourth day the opposite corner was cut off, and on the eighth day it was removed completely. The Parafilm® was removed when diameter growth was observed at the graft union.
Experimental Design and Statistical Analysis
The experiments were established under a completely randomized experimental design, with one graft as experimental unit and different replicates per treatment: 25, 10, 30, 20 and 32 from the first to the fifth experiment, respectively.
The assumptions of normality and homogeneity of variances were verified using the Proc Univariate (Shapiro Wilk) and Proc GLM (Levene Test) procedures. In each experiment, differences in graft success between phenotypic classes were identified using ANOVA with the GLM procedure of SAS (SAS Institute, 2001). Subsequently, mean comparison tests were performed with Tukey's method (P ≤ 0.05).
Grafting Success Rate Analysis
To analyze the average graft success time, the Weibull accelerated failure time model was fitted: In (T) = α + δx + σԑ; where, ln (T) = natural logarithm of the average graft success time; α, δ and σ = shape, estimation and scale parameters of the model, respectively. δ takes values of -∞|∞, therefore, this value determines the effect of the independent variable (x) on the average graft success time (Kundu et al., 2019).
Furthermore, the hazard ratio (HR) was adjusted to assess the likelihood of graft success: HR = [te^(-βx)]^(λ-1); where, β and λ are the estimator and shape parameter of the model, respectively (Pérez-Luna et al., 2020). If λ > 1, the value of HR increases, and it decreases if λ < 1 (Ghorbani et al., 2016; Zhang 2016). The model uses Dummy variables to predict HR, including censoring variables (individuals that did not experience the evaluated event -graft success) (Pérez-Luna et al., 2020). Hence, successful grafts were coded as one (1), and unsuccessful grafts (censored) were coded as zero (0). The tolerance scale and age of the donor tree for scion were coded as follows: grafts with scions from tolerant trees (0) and susceptible trees (1); grafts with scions from young trees (0) and adult trees (1). Adjustments were made using the LIFEREG procedure in SAS v9.5 (SAS Institute, 2013).
Results
In the first experiment conducted on September 8, 2020, budbreak began in the second week and ended in the fourth week after grafting. In tolerant trees, grafting success was 13 and 90 %; in susceptible trees, 60 and 80 % (Figure 2A). The graft success was affected by the source (donor plant) from which the material was obtained. The successful grafts generated an average of four to five shoots. The leaves developed to a size similar to those of adult trees and were bright green in color.
Figure 2 Average grafting and standard error in Quercus affinis grafts with A) semi-woody scions from 12-year-old trees, B) basal scions, C) semiwoody scions from adult trees and D) woody scions from adult trees. TP = tolerant phenotype, SP = susceptible phenotype. Different letters indicate statistical differences according to Tukey's test (P < 0.05).
In the second experiment on October 8, 2020, graft success differed due to the effect of the donor tree: 60 % in the tolerant and 50 % in the susceptible (Figure 2B). The first shoots were observed one month after grafting, and the last ones sprouted after two months. A particular characteristic of this type of material was the homogeneous union of the graft. The shoots were vigorous, and the leaves grew 40 % larger and wider compared to normal leaves.
Two weeks after the third experiment conducted on November 10, 2020, the scions showed a dark brown tone on the stem, which was considered non-viable, but they remained firm. The sprouting of the semiwoody scions of adult trees began one month after grafting, and the last buds sprouted after three months. The grafts that budded in the first and second months grew moderately with small-sized leaves, while the grafts that budded in the third month grew 50 % more vigorously with large and wide leaves. Graft success was 20 % and 70 % in tolerant phenotypes and 50 % and 60 % in susceptible ones (Figure 2C). The grafting season affected the graft success of Q. affinis, as grafts performed in November took longer to bud (one to three months).
In the fourth experiment, on March 4, 2021, there was only successful grafting on the grafts with scions from the susceptible tree at 40 %. Bud sprouting began from the second week after grafting and extended up to six weeks. Two to three vigorous and fast-growing shoots were obtained in each graft. This experiment had the longest shoots in the shortest time, with large leaves and light green color.
In the fifth experiment, on September 28, 2021, phenotype affected grafting. The first buds sprouted from the third week and the last ones at seven weeks. Scions of the 4TP tree had 19 % grafting on Quercus affinis rootstocks and 31 % on Q. rugosa; on the 5TP tree, 44 % on Q. affinis and 88 % on Q. rugosa. Leaves grew strong and had good size (Figure 3).
Figure 3 Average grafting and standard error of Quercus rugosa and Quercus affinis rootstock grafted with semi-woody scions from two tolerant adult trees. Different letters indicate statistical differences according to Tukey's test (P < 0.05).
Fitting the Weibull accelerated time model
A significant effect of donor tree age and tolerance scale on the time and probability of grafting was found (Table 3). When developing the accelerated failure time equation, it was estimated that grafting occurred on average at 98 and 41 days in grafts made with scions from adult and young trees, respectively. Grafting success rate was low for the two ages of the ortets, which decreased by 61 and 85 % in grafts made with adult and juvenile scions.
Table 3 Fit parameters and hazard ratio values of the accelerated Weibull time-failure model.
| Variable | Estimates | Hazard ratio (HR) | Grafting success rate [% = (HR -1) * 100] |
||
|---|---|---|---|---|---|
| α | β | Scale | |||
| Age | 6.37 | -0.86 | -0.91 | 0.15 | -85 |
| Scale | 4.27 | 0.60 | -0.96 | 3.51 | 251 |
On the other hand, it was calculated that grafting occurred on average at 50 and 92 days in grafts made with scions from susceptible and tolerant trees, respectively. Grafting success rate was estimated to be high for both types of tolerance in Q. affinis trees. Grafting success rate with scions from tolerant trees was 166 % higher than that of grafting with scions from susceptible trees.
Discussion
The damage scales, based on a series of illustrations with symptoms of the pest at different levels of intensity in the whole plant, stem, crown and shoots, are suitable for the evaluation of the damage caused and the planning of future remediation (Alfenas et al., 2009). This information allowed the rescue and propagation of tolerant individuals, which have not been affected in their growth and development by the wasp.
Grafting success rate with scions from tolerant and susceptible trees was contrasting; likewise, greater vigor and turgidity were observed in scions collected from trees tolerant to the attack of A. quercuslaurinus. Almqvist (2013) indicates that the morphology, anatomy, and physiology of both the scion and the rootstock are determining factors for grafting success.
Fast grafting success is mainly due to the correct adhesion of parenchymal tissues, which promotes early union between scion and rootstock (Pina et al., 2012). For Q. affinis, grafting occurred at two weeks with semiwoody scions from young trees (12 years old) in early autumn. The first evidence of union between scion and rootstock is the formation of callus, which functions as a weld between both organs (Castro-Garibay et al., 2017; Venturini & López, 2010). The species Q. affinis has aptitudes to be propagated by grafting given its capacity for callus generation in all the experiments developed in this study.
To obtain basal shoots of Q. affinis it is necessary to cause a wound, either in the roots or at the base of the stem, a practice that promotes the generation of auxins and, consequently, the growth of vigorous shoots with long internodes, herbaceous texture and small buds (Alfonso-Corrado et al., 2004; Cabrera-Ramírez et al., 2022). However, in the experiment, shoots appeared at approximately two months and generated few buds per scion, which reduced the number of scions available for grafting. On the other hand, the use of herbaceous scions allowed homogeneous union (callus formation in the tie area), which is possible because of the metabolic activity of scions grafted and sprouted buds (Hartmann et al., 2014).
The auxin/cytokinin ratio plays an important role in vascular regeneration within the graft union zone; when the donor plants are young trees, callus generation is higher (Goldschmidt, 2014). Quercus eduardii Trel. and Q. potosina Trel. have been observed to be able to multiply by basal shoots naturally (Alfonso-Corrado, 2004). This type of material is ontogenetically younger as was the case in the second experiment on October 8, 2020; in addition, the buds of the grafted scions remained in final dormancy, which promoted the growth of the shoots after grafting (Gómez et al., 2017).
Woody shoots from adult trees grafted in March had greater elongation. This demonstrates that scion physiology significantly influences the growth of grafts of this Quercus species. Conversely, with pines, it has been observed that scions from young trees have rapid elongation and abundant branching, while scions from older trees do not exhibit the same level of elongation and branching (Pérez-Luna et al., 2019; Velisevich et al., 2021).
In this regard, it was observed that the growth of oak grafts is influenced by the age of the donor tree, the phenological stage, and the size of the bud. Therefore, when the donor tree is closer to the peak of ontogenetic growth, the branches of the grafted scion are more elongated and abundant (Day & Greenwood, 2011). On the other hand, interspecific grafts facilitate the combination of characteristics from both the scion and the rootstock of another species to produce a plant with better performance (Loewe-Muñoz et al., 2019). This is often done primarily in fruit trees to take advantage of species resistant to pests used as rootstocks (Ullon-Chiriguaya et al., 2022).
The use of woody scions prevents obtaining good grafting succes, due to the phenological and physiological mismatch with the rootstock (Barrera-Ramírez et al., 2020; Crecente Campo & Fernández Lorenzo, 2008). This study shows the highest grafting success rate and buds with greater vigor in Q. affinis grafts with semihardwood scions from both young trees (experiment 1) and adults (experiment 5) using the apical fissure technique. Likewise, it was observed that herbaceous shoots easily lose turgidity.
The grafting season and type of scion influence grafting success in Q. affinis. This is because the buds enter a dormant phase due to decreased temperatures, which affects grafting and growth (Hibbert-Frey et al., 2010). Short photoperiods and low temperatures are the main factors influencing bud dormancy induction. This effect depends on the species and physiological age of the trees; however, it is likely that the effect is combined (Valencia-Manzo et al., 2017).
The growth in grafted shoots exhibited a high level of phenotypic variation among grafting seasons. In phenotypes with low grafting, it is advisable to perform serial grafting (obtaining scions from previously grafted trees) to have grafting success (Zaczek et al., 2006). For example, Crecente Campo and Fernández Lorenzo (2008) reported grafting success rate of 27 % in Q. robur L. with field scions (first cycle) and 69 % to 95 % between the second and tenth grafting cycles. This is possible because there is greater control of mother plants in the nursery through pruning, irrigation, and fertilization to generate scions with desirable characteristics (Castro-Garibay et al., 2022).
Conclusions
The propagation of Q. affinis individuals tolerant to A. quercuslaurinus attack is possible through homografts (from young trees: 12 years old) and heterografts (from adult trees: 35 and 40 years old) onto Q. rugosa rootstocks. Grafting success rate with scions from tolerant trees was 166 % higher than with scions from susceptible trees. The highest grafting rate in both young and adult trees was obtained in early autumn, while the most vigorous grafts were those performed in early March with scions from adult trees.
