Study on Influencing Factors and Low-Temperature Treatment of Walnut Canker Disease in Hotan County, Xinjiang

Abstract

The aim of this study was to investigate the factors affecting the occurrence of walnut canker disease and the effect of low-temperature treatment on its occurrence in Hotan County, and provide a theoretical basis for the prevention and control of the disease. The occurrence of canker disease was investigated in walnut plantations of different varieties, plant and row spacing, and diameter at breast height (DBH) from April to August of 2023 and 2024. In laboratory research, the spread area of disease spots on isolated branches of peach was studied under different temperature treatments and different treatment times. The occurrences of walnut canker for the two consecutive years were basically the same. The number of new lesions showed an upward trend in April and May and reached its highest in May. The number of new lesions tended to stabilize in June and July in Hotan County. The walnut variety with more severe disease was “Xinfeng”. The disease was most serious in plots with plant spacing and row spacing of 10 m × 7 m. The DBH of the more susceptible walnut trees was >30 cm. Low-temperature treatment and treatment time had a significant impact on the walnut canker lesion expansion area (p < 0.05). The key factor in lesion expansion was low temperature. The maximum lesion expansion area was 1.70 × 10⁴ mm² under treatment at 20 °C for 5 h. The walnut variety and the plant and row spacing were the key factors affecting the occurrence of walnut canker disease, with low temperature being the main factor affecting its spread.

1. Introduction

Walnut (Juglans regia L.) is a perennial deciduous tree belonging to the genus Juglans. It is one of the four major nuts in the world and is known as the “king of woody oil” [1,2]. The counties of Aksu, Hotan, and Kashgar are the main walnut-growing areas in Xinjiang. The pillar industry for farmers in Xinjiang is the walnut industry, whose income accounts for more than 40% of farmers’ annual economic income [3]. The walnut planting area in the Hotan area in 2022 reached 1.15 × 10⁵ hm², with 1 × 10⁵ t of fruit being produced [4]. Walnut canker disease is an increasingly serious threat to production due to planting density, variety differences, and the low-temperature climate in Xinjiang, among other factors [5]. Walnut canker disease, which is caused by the fungus Cytospora chrysosperma [6,7], is one of the major diseases affecting the healthy growth of walnut trees in Xinjiang. The incidence of walnut canker in the southern walnut cultivation region of Xinjiang can reach as high as 70%–90% [8,9]. Therefore, it is crucial to identify the key influencing factors contributing to the occurrence of walnut canker disease. Temperature has a significant effect, with its action being mostly reflected in the dual regulation of plant physiological metabolism and pathogenic microorganism activity. Experimental results have shown that low-temperature stress can significantly inhibit the activity of key enzymes in the photosynthetic system of walnut trees. Constant sun damage can lead to a deterioration in the function of the photosynthetic organs, leading to the systematic decline of plant cold resistance, thus aggravating the occurrence of walnut canker disease under this comprehensive stress effect [10]. Temperature can also affect the growth and expansion of putrefaction bacteria in the xylem, thus affecting the occurrence of putrefaction disease [11]. Investigation and research into walnut canker in the Aksu region found that the sustained low temperatures and freezing damage in winter readily led to the weakening of walnut trees, which further led to the nutritional imbalance of the tree and increasing the probability of walnut rot the following year [12]. The incidence of walnut canker disease is accompanied by low-temperature sunburn [13]. Low-temperature sunburn in the winter causes bark cracking, cambium damage, and tree weakening, which is another important cause of walnut canker disease. The degree of infection varies according to the walnut variety. Late-bearing walnuts have strong disease resistance, while early bearing walnuts have weak resistance. High-yielding varieties are also prone to infection [14]. It was found that the number of pathogenic bacteria, low temperatures, and low-temperature treatment time had significant effects on the incidence, susceptibility index, and incubation period of walnut canker [15]. Low-temperature induction can stimulate the growth of the mycelia of fungal pathogens related to canker disease and enhance the pathogenicity of these fungal mycelia [16]. It is of great significance for the monitoring and prevention of walnut canker to clarify the influencing factors of the disease in Hotan County, the main producing area of walnut in Xinjiang, and analyze the correlation between low temperatures in the winter and walnut canker. Thus, the incidence of the disease in walnut orchards of different varieties, tree ages, and cultivation methods in Hotan County was investigated in this study, and the related factors affecting occurrence were analyzed. To study the occurrence of walnut canker disease under different low temperatures and treatment times, healthy walnut branches were inoculated with walnut canker. The results provide a theoretical basis for the scientific prevention and control of walnut canker disease and benefit the healthy development of the walnut industry in Xinjiang.

2. Materials and Methods

2.1. Sample Plot Overview

Three distinct sample plots were established in Buzaq Township, Hotan County, Hotan Prefecture, and Xinjiang Uygur Autonomous Region to investigate the effects of walnut varieties, plant spacing, diameter at breast height (DBH), and intercropping methods. Sample plot 1 featured the walnut variety “Xinfeng” with a plant spacing of 6 m × 5 m and a DBH ranging from 10 to 20 cm, intercropped with corn and wheat. Sample plot 2 contained the walnut variety “Zha343”, with a plant spacing of 8 m × 6 m and DBH between 20 cm and 30 cm, also intercropped with corn and wheat. Sample plot 3 contained the “Xinfeng” walnut variety again, but with a wider plant spacing of 10 m × 7 m and DBH exceeding 30 cm, similarly intercropped with corn and wheat.

2.2. Investigation of the Influencing Factors of Walnut Canker Disease Occurrence in the Field

From April to August in 2023 and 2024, 30 walnut trees were selected in each sample plot and marked with paint. At the end of each month, new disease spots were investigated by means of a plant-by-plant survey. At the same time, temperature and humidity data in the sample plot were collected to analyze the effects of temperature, humidity, variety, DBH, and row spacing on the occurrence of walnut canker.

2.3. Effect of Different Low Temperatures and Treatment Times on the Occurrence of Walnut Canker Disease

2.3.1. Test Strains and Source of Branches

The tested strain XJAU-HT10-1 is Cytospora chrysosperma, which was selected by the research team after isolation, identification, and pathogenicity determination [17].

The inoculated branches were collected from walnut orchards during field sampling. These samples consisted of healthy, lignified 1–2-year-old walnut branches. Before inoculation, the branches were rinsed with sterile water to remove surface dust and then maintained under humid conditions for subsequent experimental use.

2.3.2. Effect of Different Low-Temperature Treatments on Occurrence of Walnut Canker Disease

The strain was inoculated on potato dextrose agar (PDA) and cultured at 25 °C until the strain covered three-quarters of the petri dish. Next, a 5 mm diameter sterile hole punch (Sangon Biotech, Shanghai, China) was used to lift the cake from the edge of the colony. The collected cake was used for subsequent inoculation. The prepared branches, cut to 20 cm lengths, were initially rinsed twice with tap water, surface-sterilized using 75% ethanol-soaked cotton, and then washed three times with sterile water before being kept moist for later use. The upper ends of the branches were sealed with paraffin, while the lower ends were cut at an angle and submerged in sterile water for maintenance under the conditions of constant temperature (25 °C), light, and humidity.

Based on a comprehensive analysis of temperature data from the sample plot between 2023 and 2024, which indicated low temperatures ranging from 5 °C to −20 °C, six treatment temperatures were selected: 5 °C, 0 °C, −5 °C, −10 °C, −15 °C, and −20 °C. The branches were exposed to these temperatures for durations of 5 h, 10 h, 15 h, and 20 h. Under sterile conditions, a 5 mm diameter sterile hole punch was used to remove the epidermis of the branches, followed by inoculation with mycelial plugs of the pathogen. Control treatments involved inoculation with blank medium. Each treatment included three replicates, with five healthy branches per replicate. Mycelial plugs were removed 24 h post-inoculation. Disease progression was monitored every 3 days, with lesion areas measured and recorded until no further expansion was observed.

2.4. Statistical Analysis

Data were organized using Microsoft Excel 2019. Following the removal of mycelial discs, the length (L) and width (W) of lesions on the branches were measured every 3 days using the cross method. The lesion area was calculated as the total elliptical area (S = πLW/4) [18,19]. One-way ANOVA was performed using SPSS 22.0 to analyze the obtained data, and a linear regression model was employed to identify the primary factors influencing lesion expansion. GraphPad Prism 10.1.2 and Origin 2022 were utilized for graphical representation and significance marking (p < 0.05).

$$\mathrm{S}=\mathrm{L}\times \mathrm{W}\times \pi /4$$

(1)

3. Results

3.1. Investigating Factors Affecting Walnut Canker Disease Occurrence

3.1.1. Influence of Temperature on the Occurrence of Walnut Canker Disease

The analysis of temperature changes in 2023 and 2024 revealed a consistent trend in the incidence of walnut canker disease. From April to May, as temperatures increased, the number of lesions rose, with the highest rate of new lesion development observed in May. Specifically, in May 2023 and 2024, newly added lesions accounted for 35.23% and 25.06% of the total lesions throughout the growth period, respectively. Between June and July, the number of lesions stabilized. The peak temperatures were recorded in July of both years (32.33 °C in 2023 and 35.48 °C in 2024), during which the number of new lesions was at its lowest, representing 10.5% and 17.58% of the total lesions throughout the growth period, respectively. In August, as temperatures began to decline, there was a resurgence in lesion development, reaching a secondary peak that accounted for 16.07% and 23.07% of the total incidence throughout the growth period (Figure 1).

3.1.2. Impact of Humidity on Walnut Canker Disease

From April to May in 2023 and 2024, the number of new lesions increased as relative humidity rose. In May 2023, both the relative humidity and the rate of new lesion development reached their respective peaks. In July of both years, the highest and lowest relative humidity levels were at their lowest points (highest relative humidity: 87.9% in 2023 and 72% in 2024; lowest relative humidity: 22.5% in 2023 and 33% in 2024), during which the number of new lesions was minimal, accounting for 10.5% and 17.58% of the total new lesions throughout the growth period, respectively. From July to August, as relative humidity increased, there was a resurgence in lesion development, reaching a secondary peak that accounted for 16.07% and 23.07% of the total incidence throughout the growth period, respectively (Figure 2).

3.1.3. Impact of Walnut Varieties on Incidence of Canker Disease

The incidence of walnut canker disease varied significantly among different walnut varieties. The walnut variety “Xinfeng” exhibited a higher total number of new lesions compared with “Zha 343”. Both varieties showed a peak in new lesion formation in May, with “Xinfeng” exhibiting up to 161 new lesions and “Zha343” exhibiting 111 new lesions. In June, the number of new lesions decreased markedly for both varieties, before increasing again in August (Figure 3).

3.1.4. Effect of Plant Spacing and Row Spacing on Occurrence of Walnut Canker Disease

The incidence of walnut canker disease in walnut plots with different plant spacing and row spacing was significantly different (p < 0.05). Specifically, the plot with a plant spacing of 10 m and row spacing of 7 m had a higher number of new lesions compared with those with a plant spacing of 6 m and row spacing of 5 m, and a plant spacing of 8 m and row spacing of 6 m. The trend in the number of new lesions over time followed a pattern of an initial increase, stabilization, and subsequent resurgence. In April, all three plots reached their peak in new lesion formation, with the 10 m (plant spacing) × 7 m (row spacing) plot exhibiting 165 new lesions, while 113 and 122 new lesions were recorded for the 6 m × 5 m and 8 m × 6 m plots, respectively. The number of new lesions decreased markedly in June before increasing again in July (Figure 4).

3.1.5. Effect of Different DBHs on Walnut Canker Disease

The incidence of walnut canker disease in plots with different DBH classes exhibited significant differences (p < 0.05) across all months. Specifically, walnut trees with DBH > 30 cm consistently developed more new lesions compared with those in 10–20 cm and 20–30 cm DBH classes. In April, new lesions peaked at 177 for >30 cm DBH trees and 113 for 10–20 cm DBH trees. The 20–30 cm DBH class reached its maximum (114 lesions) in May. All three DBH categories showed decreased lesion counts in June followed by a resurgence in July. These findings suggest that trees with larger DBHs are more susceptible to walnut canker disease (Figure 5).

3.2. Effects of Low-Temperature Treatments on the Expansion of Walnut Canker Disease Lesions

3.2.1. Dynamics of Low-Temperature Gradients on Walnut Canker Lesion Expansion

The effects of different low temperatures on the expansion area of walnut canker lesions were significantly different (p < 0.05). After 10 h of treatment at 5 °C and −15 °C, the lesion expansion area was significantly affected (p < 0.05), reaching the maximum values of 3.65 × 10² mm² and 3.66 × 10² mm², respectively; after 20 h of treatment at 0 °C, the lesion expansion area was significantly affected (p < 0.05), with the maximum value reaching 4.94 × 10³ mm²; after 15 h of treatment at −5 °C and −10 °C, the lesion expansion area was significantly affected (p < 0.05), reaching the maximum values of 4.71 × 10³ mm² and 8.05 × 10² mm², respectively; after 5 h of treatment at −20 °C, the lesion expansion area was significantly affected (p < 0.05), with the maximum value reaching 1.70 × 10⁴ mm² (Figure 6).

3.2.2. Effects of Low Temperature on Lesion Expansion at Different Treatment Times

Different treatment durations significantly influenced the lesion expansion area in walnut canker (p < 0.05). Specifically, the maximum lesion expansion area of 1.70 × 10⁴ mm² was observed after a 5 h exposure to −20 °C. The second largest expansion area, 4.71 × 10³ mm², occurred following a 15 h treatment at −5 °C. Conversely, treatments at −15 °C for 5, 15, and 20 h resulted in the smallest lesion expansion areas among all conditions tested. Additionally, the least impact on lesion expansion was noted after a 10 h treatment at −10 °C (Figure 7).

3.2.3. Correlation Analysis of the Expansion Area of Walnut Canker Lesions Under Different Low-Temperature Treatments

To investigate the influence of different treatment times (H) and temperatures (T) on the expansion area of walnut canker lesions (Y), a multiple regression analysis was conducted, yielding the following model: Y = −99.918 − 381.582T + 50.923H. The coefficient of determination (R² = 0.366) indicates a significant correlation between lesion expansion and the investigated factors (p < 0.01). Specifically, low temperature had a significant negative effect on lesion expansion (B = −381.582, p < 0.001), meaning that, for each unit increase in temperature, the lesion expansion area decreased by 381.582 units. In contrast, the effect of time on lesion expansion was not statistically significant (B = 50.923, p = 0.584), suggesting that, within the current time scale, the direct impact of time on lesion expansion is minimal.

4. Discussion

Based on a two-year investigation (2023–2024) of the influencing factors of walnut canker in Hotan County, it was observed that the disease began to spread in April with increasing temperature and humidity, reaching its peak in May. The number of new lesions started to decline in June, but increased again in July and August. This pattern aligns with the findings of Chen [20], indicating that temperature significantly influences the infection of conidia and the growth of xylem hyphae. Furthermore, temperature modulates fungal spore germination, a critical factor potentially exacerbating disease severity through enhanced pathogenic development [21]. Different walnut varieties exhibit varying degrees of susceptibility to canker, likely due to the frequent occurrence of late-spring cold snaps and late frost damage in northern China. These climatic events affect the frost resistance of different walnut varieties, which, in turn, influences their susceptibility to walnut canker [10]. In this study, the walnut variety “Xinfeng” in Hotan County was found to be more susceptible to walnut canker than “Zha343”, possibly due to differences in frost resistance. Varietal differences in walnut cultivars significantly influence stress tolerance phenotypes, potentially inducing corresponding divergence in disease-resistance capabilities [22,23]. Further research is warranted to explore this relationship. The incidence of walnut canker was higher in plots with plant spacing and row spacing of 10 m × 7 m compared with plots with plan spacing and row spacing of 6 m × 5 m and 8 m × 6 m. The findings of this study align with those of previous research on wheat Fusarium head blight (FHB). Expanded row spacing is associated with elevated FHB incidence and mycotoxin contamination, ultimately leading to yield reduction. However, the combined application of high-nitrogen fertilization and fungicide demonstrates economic viability only under conditions of low FHB risk and elevated grain market prices [24]. Additionally, the incidence of new walnut canker spots in plots containing walnuts with a DBH greater than 30 cm was consistently higher compared with plots with walnuts with a DBH of 10–20 cm and 20–30 cm. Research has established a significant positive correlation between tree age and DBH. During ontogenetic development, trees exhibit a progressive DBH increment. Specimens with larger DBH demonstrated enhanced physiological resilience, potentially attributable to prolonged vegetative growth periods, delayed xylem maturation, modified dormancy regulation mechanisms, and altered cold sensitivity responses [25,26].

Low-temperature treatment experiments confirmed that both low temperatures and treatment duration significantly impact the expansion area of walnut canker lesions. Low temperature is a critical environmental factor influencing lesion expansion, with its effect intensity negatively correlated with the temperature gradient. Specifically, when the temperature drops to −20 °C and is maintained for 5 h, the lesion expansion area reaches its maximum value. The conclusion reached in this study regarding the promotion of walnut canker disease by low temperatures is consistent with that of Yue [15]. Studies have found that, when the temperature is lower than −15 °C, the cork layer and cortex of the branches can be destroyed, thus increasing the sensitivity of apple branches to the infection of canker bacteria [27,28]. Disease severity in apple leaves and branches was significantly increased when the pathogen was exposed to −10 °C to 10 °C for more than 18 h, and the ratio of lesion area, length, and lesion frequency was higher than those at 25 °C [29]. In the present study, it was also found that the maximum area of walnut canker spot expansion was observed when treated at a temperature of −20 °C for 5 h. A study on the expansion of V. mali lesions across seasons revealed that the time required for visible spot formation varied with inoculation timing [30]. After winter dormancy and cold stress, trees weakened, facilitating faster lesion expansion—consistent with our findings.

5. Conclusions

In Hotan County, the walnut variety Xinfeng exhibited higher disease susceptibility as temperatures and humidity rose from April to May. Disease severity was particularly pronounced in sample plots with a planting spacing of 10 m × 7 m and trees exceeding 30 cm in diameter at breast height (DBH). Among environmental factors, low temperature was identified as the key driver of walnut canker lesion expansion in Xinjiang, showing the strongest correlation with disease progression. Therefore, we recommend prioritizing disease-resistant cultivars, optimizing planting density, enhancing the management of large-DBH trees, and integrating low-temperature early warning systems into disease prevention strategies.