A New Source of Inoculum for Stemphylium vesicarium: Consequences for the Management of Brown Spot of Pear

Abstract

Brown spot of pear (BSP), caused by Stemphylium vesicarium, is one of the most dangerous pear fungal diseases, being responsible for huge losses in production. Currently, in order to increase its containment, chemical control is implemented in conjunction with agronomic techniques able to reduce BSP inoculum sources (e.g., orchard grass sanitation, litter removal or application of biocontrol agents). Regardless, despite the introduction of agronomic practices, the complete control of the disease is still rarely possible, which suggests that other sources of S. vesicarium inoculum that are currently neglected may be involved. The aim of this study is to investigate the possible wintering forms of Pleospora allii/S. vesicarium on pear wood and whether any spore-forming productions (conidial or ascosporic) might infect the green tissues of the plant in the following spring. Symptomatic fragments of woody tissue from a commercial pear orchard (in Ferrara, Emilia-Romagna, Italy) with a high BSP pressure (~40% incidence) were analysed. The results prove that pseudothecia and the maturation of ascospores of P. allii also develop on one-year-old branch cankers of pear trees, thus representing an additional source of inoculum. In conclusion, the pruning of affected branches and removal of relative residues should be preventatively performed in order to improve BSP management and control.

1. Introduction

Brown spot of pear (BSP), caused by Stemphylium vesicarium (Wallr.) E. Simmons [1], is one of the most serious fungal diseases affecting pear production. It can cause significant losses of production in most European pear growing areas. BSP was first reported in the mid-1970s in Emilia-Romagna (Italy) on cv Abate Fetel [2]. Subsequently, the disease rapidly spread in France, Spain, Portugal, Holland and Belgium [3]. Symptoms can be detected from flowering to harvesting and consist of necrotic lesions on leaves, twigs, petioles and especially on fruits, more than 90% of which can be affected [4]. On fruit, the spots are initially circular and brown, sometimes surrounded by a red halo, and located in the equatorial area or in the calyx area of the fruit. Subsequently, they can expand due to the invasion of saprophytic fungi, like Alternaria spp., also leading to fruit rot [5]. Disease intensity depends on inoculum levels and environmental conditions [6]. Regardless, whatever the level of the disease, affected fruits are not marketable.

Like other fungal diseases, BSP is characterized by two forms of inoculum: (i) the sexual type consists of pseudothecia and ascospores of Pleospora allii (Rabenhorst) Cesati & de Notaris and (ii) the asexual one of conidia of S. vesicarium. The sexual phase is responsible for the conservation of the fungus and takes place during autumn and winter on orchard debris [6]. However, the asexual stage is the main cause of infections occurring on leaves and fruits and causing huge losses in production. In contrast with the sexual stage, the asexual one takes place during spring and summer [3].

The cycle of the disease is extremely complex due to the saprophytic capacity of S. vesicarium to colonize several plant herb species common in pear orchard grass [7]. Pseudothecia were observed mainly on pear fruit and leaf litter [8], on Poaceae debris and Fabaceae debris species [7]. Their growth depends on the presence of high relative humidity (>98%) with optimum temperatures ranging from 10 to 15 °C [8]. The first mature pseudothecia may be detectable between December and February [4]. The release of ascospores from pseudothecia occurs in the presence of heavy dew and rainy events, usually from February to early June and between August and October [4,9]. The main role of the ascospores dispersed in the environment is to colonize plant residues, which will act as the primary source for conidium production during the pear growing season [9]. As already anticipated, conidia (asexual spores) are the major spores responsible for infections on pear, causing the most economically significant damages. Indeed, although the ascospores of P. allii have been shown to infect pear leaves and fruit [4], the period of when symptoms appear seems to be unrelated to the period of the greatest release of ascospores [3]. In Italy, the first symptoms are usually detectable starting from late spring, when ascospore flight is usually low; thus, the peak of ascosporic flight occurs long before the onset of disease symptoms [10]. Therefore, it is conceivable that ascospores have little influence on epidemic development in late spring and summer. Most likely, the main role of ascospores is the saprophytic colonization of plant herbs with the consequent production of conidia, causing infections during the pear growing period. In fact, the study of Rossi [7] has demonstrated the abilities of the conidia of S. vesicarium to colonize the dead tissues of different herbaceous plants growing in pear orchard lawns (both grasses and dicotyledons); to produce conidia in abundance (in particular on Festuca ovina and F. rubra) and to overwinter the pseudothecia of its teleomorph P. allii on pear leaves, grasses and white clover.

BSP management is extremely expensive in terms of treatments and costs. Orchards characterized by a high incidence of the disease may require 12 to 25 fungicide applications to obtain a low level of disease [6]. Nevertheless, in recent years in Italy, the disease pressure increased to such an extent that plant protection products have not been able to protect plants as effectively as in the past. In order to improve the efficacy of BSP management, further agronomic techniques have been introduced. The aim is to reduce the inoculum of the fungus in leaf litter with sanitation treatments like leaf litter removal, tillage or the use of hydrated lime or biocontrol agents [6,10,11]. The main strategy adopted during winter is removing leaves from litter or even spontaneous plants [6] or carrying out tillage on the entire surface of the pear orchard. Tilling soil is a more effective strategy than simply removing leaf debris since the inoculum sources of P. allii/S. vesicarium are weeds, especially those of the Festuca genus [7]. However, this strategy has a negative effect of eliminating the load-bearing capacity of soil, making the transiting of tractors more difficult. Biocontrol methods consist of the use of different biocontrol agents such as Trichoderma spp., Bacillus subtilis and Pseudomonas fluorescens [10,11,12]. These organisms are applied directly to litter during spring, and through their parasitization capacity, space, nutrient competition and antibiosis effects, they cause a reduction in P. allii/S. vesicarium inoculum sources.

The introduction of these new strategies for BSP management has led to a reduction in the disease intensity but not to the complete control of it [13]. This suggests that S. vesicarium may have other sources of inoculum; therefore, further investigations are needed.

To the best of our knowledge, pruning branches characterized by cankers has not yet been examined as a possible strategy to reduce inoculum sources. For this reason, the aim of this work is to investigate whether BSP cankers on wood could represent an additional source of inoculum.

2. Materials and Methods

2.1. Experimental Site

This experiment was conducted in a commercial orchard of Pyrus communis cv Abate Fetel, characterized by a high BSP pressure in the 2022 growing season. The orchard is located in San Bartolomeo in Bosco, Ferrara, Emilia-Romagna (44°42′58.6′′ N 11°35′50.1′′ E), in the Po valley, north of Italy. Twig canker incidence was estimated to be approximately 40%.

2.2. Description of Experiment

At the end of September 2022, severe twig canker symptoms (Figure 1a,b) were observed on one-year pear shoots. Symptomatic shoots were collected and taken to the phytopathological mycology laboratory of “Servizio Fitosanitario E-R”, a plant protection service located in Emilia-Romagna (Italy). Symptomatic tissue fragments were picked up from the shoot lesion and then disinfected on a surface with NaOCl 1% for 3 min, rinsed 3 times with sterile water, dried, transferred onto potato dextrose agar (PDA) and, finally, incubated in the dark for 5 days at 27 °C. The grown fungal colonies and conidia were identified as S. vesicarium according to Ellis [1].

After the confirmation of the mycological analysis, two theses were considered for the subsequent analyses. The first concerned fifty symptomatic twigs marked in the field, and the second concerned another fifty twigs that were pruned, removed and placed on the ground to reproduce a litter.

To study the evolution of twig cankers, monthly assessments were performed on five symptomatic branches for each observed thesis, evaluating the presence of S. vesicarium conidia or ascospores contained in the pseudothecia of P. allii.

After the confirmation of ascospore development on twig cankers, their ability to reproduce symptoms on young pear leaves cv Abate Fetel, using a detached pear leaf assay [12], was also investigated. In January, many twig cankers with forming pseudothecia were collected in the field and stored at 4 °C in a refrigerating room. At the beginning of the new season (April), young shoots with leaves were collected on several asymptomatic Abate Fetel pear trees from an orchard that had not undergone any treatment with fungicides. The samples were placed in a humid chamber at 5–7 °C and transported to the E-R plant protection service. The inoculum was obtained by isolating ascospores from mature pseudothecia, which were picked up from the plant material collected in January and stored in the refrigerating room. The pseudothecia were transferred using thin sterilized forceps onto a microscope slide. Then, by applying pressure with the forceps, the walls of the pseudothecia were broken to allow the asci with the ascospores to come out. A visual analysis was performed with a microscope (N300TL, Eurotek Orma, Milan, Italy) to confirm the absence of S. vesicarium conidia. Pseudothecia were collected using a Pasteur pipette and deposited in sterile water. The ascospore suspension of 10⁶ ascospores of P. allii was calculated using a haemacytometer chamber. Shoots and leaves were inoculated with 4 drops of ascosporic suspension deposited with a Pasteur pipette. A non-inoculated test using sterile water was performed. Inoculated leaves and shoots were placed in plastic trays containing one layer of sterile filter paper moistened with sterile water, covered by a polyethylene bag and incubated at 25 °C with a photoperiod of 16 h of light. The experiment consisted of 2 replicates, each composed of 1 young shoot and 5 leaves.

After 5 days of incubation, a visual assessment was carried out to detect the presence of symptoms.

Fisher’s exact test was performed to test the null hypothesis that no nonrandom associations exist between the two categorical variables, inoculated/symptomatic, against the alternative hypothesis that there is a nonrandom association between the variables. Statistical analysis was performed with Matlab^© (MathWorks, Natick, MA, USA). p-value < 0.001 was considered significant.

3. Results

To study the evolution of twig cankers, the presence of the conidia of S. vesicarium or ascospores contained in the pseudothecia of P. allii was evaluated through monthly assessments in five symptomatic twigs for each thesis observed (

Table 1. The results of the monthly observations on the development of pseudothecia on twig cankers placed on the litter and left on the plants.

Assessment N.	Twig Cankers in the Litter	Twig Cankers on the Plant
1. October 2022	No pseudothecia	No pseudothecia
2. November 2022	Paraphysis formation	No pseudothecia
3. December 2022	Presence of asci and ascospores	Paraphysis formation
4. January 2023	Presence of asci and ascospores	Presence of asci and ascospores

).

In the first survey carried out in October 2022, no changes to the cankers were observed. Starting from the second evaluation, carried out in November 2022, formant pseudothecia were identified only on the twig cankers left in the litter (Figure 2).

Pseudoparaphyses that fill the lumen were detected under a microscope at 20× magnification (Figure 3). No evolution was detected on the twig cankers left on the plants.

The third assessment carried out in December outlined mature pseudothecia on twig cankers left in the orchard litter with fully developed ascospores inside, ready to be dispersed in the case of rain (Figure 4). The ascospore’s morphological characteristics are attributable to the teleomorph P. allii [14].

On the other hand, during the same survey, the first pseudothecia with still-forming asci were found in the twig cankers left on the plants.

Only the fourth evaluation, carried out in January, pointed out the first mature ascospores of P. allii in the pseudothecia (Figure 5a,b) from the cankers left on the pear tree branches (Figure 6).

Finally, the ability of the ascospores of P. allii developed on twig cankers to reproduce symptoms on young pear leaves cv Abate Fetel was investigated. This analysis was performed through a detached pear leaf assay [12]. After 5 days of incubation, a visual assessment was carried out to detect the presence of symptoms. All the inoculated shoots and leaves showed typical BSP necrosis, usually detected in the field (Figure 7). No symptoms were detected on the water-inoculated samples. In replicate 1, 22 drops out of 24 of the ascosporic suspension showed BSP symptoms, while in replicate 2, 21 drops out of 24 showed BSP symptoms (

Table 2. Number of shoots and leaves inoculated with 4 drops of ascosporic suspension of P. allii at concentration of 10³ ascospores and number of drops of ascosporic suspension showing brown spot of pear (BSP) symptoms after 7 days of incubation at 25 °C with 16 h photoperiod of light.

Thesis	Number of Shoots Inoculated	Number of Leaves Inoculated	Number of Ascosporic Drops with Visible Symptoms	Number of Ascosporic Drops without Symptoms
Shoot and leaves inoculated—replicate 1	1	5	22	2
Non-inoculated check—replicate 1	1	5	0	24
Shoot and leaves inoculated—replicate 2	1	5	21	3
Non-inoculated check—replicate 2	1	5	0	24

). The results of Fisher’s exact test prove that there is a statistically significant association between inoculation and the appearance of symptoms (p-value < 0.001).

Symptomatic tissues were disinfected on the surface with NaOCl 1% for 3 min, rinsed three times with sterile water, dried, transferred onto potato dextrose agar (PDA) and, finally, incubated in the dark for 5 days at 27 °C. The morphological evaluation of the fungal colonies and grown conidia performed according to Ellis [1] confirmed the re-isolation of S. vesicarium.

4. Discussion

To the best of our knowledge, this is the first report describing the development of the pseudothecia and the maturation of the ascospores of P. allii on one-year-old branch cankers on a pear tree cv Abate Fetel. Until now, the pseudothecia of P. allii on pear trees had only been observed on symptomatic fruits and leaves present in leaf litter [7,8].

The results of this work show that the ascospores of P. allii released by pseudothecia on one-year-old twig cankers have the ability to infect young pear tissues, thus confirming that branches affected by BSP constitute an additional and ignored source of inoculum. Indeed, regardless of the plant material on which the pseudothecia develop, the ascospores of P. allii show their ability to infect pear shoots and leaves. According to Llorente [4], the first pseudothecia were observed starting from November, while Rossi et al. [9] described the presence of ascospores already in August. This result probably reflects the ubiquity of P. allii and the different development rates of pseudothecia and ascospores, which may vary based on the host plant species and the type of tissue.

Furthermore, the pseudothecia present in twig cankers left in the orchard litter develop faster than those present in the cankers of one-year-old branches of viable trees. This result could be explained by the high relative humidity required for the growth of pseudothecia [8]. Indeed, the twig cankers present in the litter likely received a greater level of humidity than the cankers of the one-year-old branches left on the pear trees.

This new information on the sources of inoculum of P. allii has important implications for the agronomic management of the disease. As demonstrated, BSP cankers on branches constitute an additional source of inoculum during the spring (April to May), which should not be ignored in commercial pear production. The elimination of all one-year-old branches showing cankers and the removal of all pruning debris from the orchard are necessary measures to be included in the complex control strategy against BSP. This could also lead to a reduction in the number of necessary treatments and, more generally, to a better efficacy of the disease control strategy.

Moreover, the presence of small cankers produced on scions in nursery plant production may be considered as a possible pathway for the dissemination of the pathogen in BSP-free pear production areas. Fungicide treatments, used during the production of plant propagating materials, may also contribute to disseminating a resistant population of S. vesicarium to some active ingredients, thus leading to a further spreading of resistant strains through sexual mating.

In conclusion, this study represents a step towards obtaining a better understanding of BSP biology and, at the same time, towards obtaining an agroecological reduction in BSP inoculum sources. Further insights are needed to improve the biological knowledge of BSP, for instance, through controlled experiments on whole plants, in line with Koch’s postulates [15] and especially through analysing the mechanisms characterizing pseudothecium maturation in different environmental conditions, including the type of host tissue, and their role on disease intensity. The novel information introduced in this work can lead to the adaptation of BSP sanitation measures, contributing to understanding the reasons why the efficacy of control is incomplete, thus innovating pear orchard management, making it more sustainable from the economic and ecological points of view.