Fire blight, a bacterial disease of pome fruit trees, is becoming more damaging due to warmer and wetter weather patterns in spring (Menzel et al., 2006; Keenan et al., 2014; Ault et al., 2015; Griffiths, 2021). Particularly, the Northeastern United States (U.S.) has experienced an increase in 0.09°C in temperature and 10-mm in precipitation per decade, with a 70% rise in precipitation as heavy rain events (Groisman et al., 2013; Kunkel et al., 2013). In the last 6 years, an alarming trend was recorded in the Northeastern and Mid-Atlantic U.S. of more years with springs characterized by an extremely warm and wet weather that facilitates continuous blocks of 3 – 7 days with high fire blight infection potential according to developed prediction models, often reaching very high values in just a few days, and allowing 3 to 9 fire blight infection events during apple bloom (Philion and Trapman, 2011; Cornell University, 2016; RIMpro-Erwinia, 2016). It is predicted that more fire blight epidemics in the Mid-Atlantic, Northeastern, and Northwestern U.S. are poised to occur with the increasingly wetter and warmer climate (Frumhoff et al., 2007; Hayhoe et al., 2007; CBC News, 2014; CBC News, 2016; Robbins, 2019). For example, it is highly probable that protracting epidemics like the ones in Washington state from 2017 to 2019, could repeat. Fire blight removal in this state during 2017 alone cost pome fruit growers $9 million on 21,450 ha, while 78.5 ha of pear and 121 ha of apple were removed (DuPont 2021, personal communication). Cost of removal by pruning ranged from $66.7 to $2,135 per ha. The fire blight outbreaks continued in 2018 and 2019, with cumulative effect of losses (DuPont 2021, personal communication). In New York state (henceforth “NY”), sudden fire blight epidemics in 2000 and 2008 (Douglas, 2022) led to 50% apple tree losses in young, recently planted orchards (Breth 2008). In 2016, severe regional fire blight epidemic in northern NY caused more than $16 million in damage (Aćimović and Meredith, 2017). High- and medium-density apple orchards have lost 2,500 trees per farm on average, due to fire blight cankers developing on apple tree central leaders and rootstocks (Figure 1). Tree losses continued in 2017 because of latent (asymptomatic) fire blight infections of rootstocks (Aćimović and Meredith, 2017).

The life cycle of fire blight pathogen, Erwinia amylovora (Burrill; Winslow et al., 1920) starts by survival in apple wood bark around fire blight canker edges and below the visible canker necrosis (Santander et al., 2019, Santander et al., 2022b). In spring, E. amylovora emerges on the surface of cankers and can be disseminated by insects, rain, or wind to flowers and shoots. Under favorable warm and wet weather conditions during bloom: (I) ≥18.3°C average daily temperature; (II) 198 degree hours >18.3°C accumulated from first open flowers; and (III) ≥ 0.25 mm rain or heavy wetting dew or ≥0.25 mm rain the previous day, which facilitate E. amylovora population growth on flower stigma and washing of these colonies to nectar glands, flower and shoot infections establish and lead to blossom and shoot blight (Turechek and Biggs, 2015). New infections of succulent tissue allow the bacterium to move via xylem and parenchyma and invade wood tissues to develop fire blight cankers. E. amylovora residual cells can asymptomatically migrate far beyond the visible symptoms in the tree canopy (Steiner, 2000) and reach the tree rootstock (Momol et al., 1998; Vanneste and Eden-Green, 2000). This systemic cell transfer occurs via xylem of the central leader and trunk to the graft union (Momol et al., 1998; Norelli et al., 2003a). An alternative pathway for the pathogen to invade the rootstock are infections of rootstock suckers growing around the base of the tree. Crown suckers emerge in the area immediately surrounding the rootstock trunk base (Figure 1C, red arrows), while the root suckers develop from roots further away from the trunk (Figure 1C, blue arrow) (Utah State University - Extension, 2020; Utah State University, 2022).

Depending on the level of the rootstock’s genetic fire blight susceptibility, infections with E. amylovora can express as development of cankers on the bark, often referred to as rootstock blight, which may girdle the rootstock stem or trunk base and kill the tree (Figure 2) or remain asymptomatic as latent infections (Norelli et al., 2001; Aldwinckle et al., 2004; Johnson and Temple, 2016). There are differences in rootstock susceptibility to fire blight. Very susceptible to fire blight are Malling series M.26 and M.9 and its subclones (Nic29, T337, Pajam 2), tolerant or moderately resistant to fire blight are M.7, and Budagovskij series B.9 and B.118, and fire blight resistant are Geneva series G.11, G.41, G.202, G.214, G. 890, G.935, G.969 and similar (Wertheim, 1998; Norelli et al., 2001; Norelli et al., 2003b; Aldwinckle et al., 2004; WSU, 2022).

In high density apple orchards, which hold between 1,200 – 5,000 trees/ha, fire blight cankers have much higher chances to quickly develop on the tree stem and rootstock due to smaller tree sizes, narrow i.e., columnar tree training systems, and short bearing limbs. These traits allow E. amylovora to quickly migrate from infected flowers and shoots to the tree stem (Aćimović et al., 2021) and cause high incidence of cankers on the central leader and rootstock. Rootstock cankers are often overlooked by growers as they are close to or below the soil line and difficult to discern from healthy bark, sometimes due to soil particles splash- or dust-covering the rootstock. Additionally, asymptomatic rootstock infections by E. amylovora are difficult to detect using classic or molecular methods as there are no clues where the samples for analysis should be collected. They often lead to surprise tree collapse and death in the current and the year following an outbreak, especially after successful aerial symptom removal by summer and winter pruning. Even if cankers are visible on the rootstock, they are particularly hard to associate by growers with the previous blight removal from the tree canopy because they often express with time delay in relation to blossom and shoot blight. This time delay leads growers to think the cause is different because they believe that fire blight was successfully and completely removed by pruning. Despite the many aspects of rootstock blight being unclear, there are limited studies recording the extent of rootstock blight damage and detecting and managing the asymptomatic infections in high density orchards (Momol et al., 1998; Norelli et al., 2001; Aldwinckle et al., 2004; Johnson and Temple, 2016).

Simultaneously with fire blight damage rising, high-density farms have caught the national attention with the syndrome of bearing apple trees abruptly dying during the summer, which was named Rapid Apple Decline (RAD) or Sudden Apple Death (SAD) (Stokstad, 2019). Necrosis and wood decay expanding from bark or vascular cambium into the heartwood were observed mostly on the rootstock wood, below the graft union of RAD/SAD affected apple trees (Singh et al., 2019). This work indicated that rootstock is the starting point of this syndrome. Diagnosing the cause(s) of rapid apple decline, that is markedly different in expression from fire blight, quickly became important and studies are underway to determine potential causes of this syndrome (Liu et al., 2018; Singh et al., 2019; Gruber, 2022). Tree decline has led the tree fruit experts to analyze weather data and postulate that extremely cold winter followed by drought in 2015 – 2016 contributed to weakening of the trees and making them more susceptible to a variety of stresses (Singh et al., 2019). However, by far, fire blight has historically been the top cause of apple tree death in comparison to other causes and was ranked as the number one IPM Research and Extension Priority for 7 consecutive years (2014-2020) by the Tree Fruit IPM Working Group at the Northeastern IPM Center (Tree Fruit IPM Working Group, 2020).

Diagnosing that trunk and rootstock cankers are caused by E. amylovora is a top challenge for apple growers because visually they can be misdiagnosed with cankers caused by the less frequent Oomycete and fungal pathogens like Phytophthora spp., Botryosphaeria dothidea, B. obtusa, Sclerotium delphinii, and Valsa mali which cause Phytophthora crown, collar and root rot; white rot of fruit and wood canker; black rot of fruit and apple wood canker; Southern blight of apple; and Valsa canker, respectively (Aycock 1966; Sutton et al., 2014; Peter, 2020; Yeonghyeon et al., 2022). What is more alarming is that most growers do not see the economic impact of fire blight cankers until apple trees start to decline and die because they are obscured by tree canopy, are large in numbers, and hard to see. It is impossible to detect all cankers on a single tree by a human eye (Aćimović et al., 2014). Due to a large number of tall, narrow trees per acre, more fire blight cankers are likely to be overlooked in high-density orchards and carryover into the next growing season perpetuating fire blight infections (Aćimović et al., 2014; Aćimović et al., 2021). The result is a recurring problem for growers to reliably determine which apple trees are infected and must be removed, signifying the practical importance of detecting E. amylovora as the cause of rootstock cankers.

With the rising impact of trunk and rootstock cankers causing tree top and whole tree death in high-density orchards, a need for rapid but cheap molecular detection for E. amylovora, bypassing pathogen culturing, increased so that the positive diagnostic result can inform tree removal and help orchard insurance claims (Courtney, 2020; USDA, 2020a; USDA, 2020b). In this work, the first goal was to use PCR test to detect E. amylovora in apple rootstocks on commercial farms affected by fire blight and deliver the results to growers to timely remove whole trees with both latent infections and visible canker(s). The second goal was to determine if E. amylovora can be detected in the trees with healthy looking, canker-free rootstocks, that are closest to the central tree with visible rootstock canker(s), thus revealing asymptomatic infections. The central trees with rootstock canker(s) were considered in need of being removed, while the trees surrounding the central tree were considered as the edge trees with potential latent rootstock infections. Both tree types comprised one infection focus. Besides canker(s) on the rootstock, the central trees often had fire blight strikes in the crown and sometimes were completely blighted or dead. The results of PCR detection on the edge trees would reveal the probability for latent E. amylovora infections and their spatial distribution. The above-described infection foci were purposely chosen because they are positioned on the rim of large groups of severely infected or dead trees and are the most difficult to decide for removal by growers. Only the dead or the trees with severely cankered rootstock, trunk, and/or central leader in these large groups are removed in fire blight eradication or replanting efforts. Therefore, knowledge of E. amylovora asymptomatic infections in the foci edge trees would serve as valuable information to growers by showing the need to expand tree removal extent beyond dead and the trees with cankered rootstocks. This would improve the efficacy of efforts to eradicate E. amylovora from the orchards timely and thoroughly.

Two groups of data are presented for the same monitored infection foci. The first group of data in the subtitle 3.1 below and Figure 4 shows the pathogen detection and disease rating parameters that were monitored only on the edge trees in the infection foci: PCR detection of E. amylovora on rootstock (positive; negative), rootstock canker incidence, healthy rootstock percent, dead and alive tree percentages. The second group of data in the subtitle 3.2 below and Figure 5 presents the action by farm staff in terms of the whole tree removal based on the PCR detection data in rootstocks from Figure 4A and encompasses all the trees in the infection foci, both central and the edge trees.

Apple rootstock infections and cankers from fire blight are economically important but poorly investigated part of E. amylovora life cycle. After natural epidemic in 2016, multiple infection foci in seven orchards planted on either M.9 or M.26 rootstock were selected. Each focus consisted of one central tree with visible rootstock canker(s) and the closest surrounding trees to it with visually healthy-looking rootstocks, or the edge trees. Such foci are usually positioned at the rim(s) of the large groups of trees dead from fire blight and present a problem for removal of infected trees due to latent E. amylovora infections in their rootstock and subsequent delayed tree death. Around 30% of latent E. amylovora infections were detected in the edge tree rootstocks in the fall of the epidemic year. One year later, fire blight cankers on the edge tree rootstocks (35%) expressed with delay, during the growing season following an epidemic. These rootstock cankers led to death of up to 35% of the edge trees. This and a few new tree deaths at the end of the experiment indicated that the zone of tree removal should be expanded beyond the borders of already dead groups of trees first killed in the epidemic, and should be conducted in multiple cycles. Such zone expansion, which should include the edge trees around the rootstock cankered trees, would ensure removal of the most or all the infected apple trees in less returns to the orchard.

As reported by Russo et al. (2007), in the last 10 years planting of high-density apple orchards in the U.S. still continues to strongly depend on the widely available M.9 rootstock. M.26 rootstock was also used in high-density systems but is mostly reserved for less vigorous cultivars. In stool beds, M.9 and M.26 rootstock “mother plants” are much more productive in growing rootstock liners, i.e., the individual shoots that will become rootstock plants, in comparison to the less liner-productive Geneva series rootstocks. Thus, for faster profitability, nurseries can grow larger quantities of M.9 and M.26 rootstocks in a short time and use them for grafting scions i.e. producing apple saplings. However, M.9 and M.26 are extremely susceptible to fire blight and in years with severe natural fire blight infection pressure, apple tree mortality greater than 50% and 60% is often recorded when orchards are planted on M.9 and M.26 rootstocks, respectively (Ferree et al., 2002; Norelli et al., 2003a; Robinson et al., 2007). In contrast, the resistant Geneva series rootstocks, G.16 and G.30, were found to have 70% less rootstock blight-related tree mortality in comparison to M.26 and M.9 in both natural and inoculated field trials (Norelli et al., 2003a). The fire blight susceptibility of M.9 and M.26 rootstocks reduces apple orchard profitability and disrupts production continuity in high-density plantings where initial costs for establishment are substantial. Higher capacity of production of fire blight-resistant rootstocks and thus their wider use are both necessary to reduce grower reliance on M.9 and M.26.

E. amylovora was not detected with PCR in the edge tree rootstocks in fall 2017. The positive PCR detection of E. amylovora in asymptomatic rootstocks in fall 2016 was significantly offset in time from the detection of cankers on rootstocks and tree mortality in fall 2017. In fall 2017, the concentration of E. amylovora in rootstocks might have declined below the limit of detection of PCR (EPPO, 2013; Ham et al., 2022). The 10.1% – 28.5% higher rootstock canker incidence in fall 2017, in comparison to the precent of E. amylovora detections with PCR in fall 2016, aligns well with the faster death of susceptible host wood from cankers and lower linked incidence of E. amylovora-positive cankers on their branches (Santander et al., 2022b). Viability digital PCR in this work showed that more E. amylovora cells die in cankers on highly susceptible pome fruit tree hosts due to large extent of damage on branches and faster tissue death, thus causing lower detection rates of E. amylovora-positive cankers. Santander et al. (2022b) also demonstrated that in fall, E. amylovora canker populations on highly susceptible Asian pear cultivar ‘Shinko’ declined to 10⁵ – 10⁶ CFU/g of canker in fall and down to non-detectable levels in winter. Thus, the high susceptibility of wood tissues to fire blight could lead to faster wood death and decline in E. amylovora concentrations in cankers to the levels below the detection limit of PCR. Fire blight rootstocks M.26 and M.9 in the current study, are rated as very susceptible to fire blight (Wertheim, 1998; Norelli et al., 2001; Aldwinckle et al., 2004; WSU, 2022). Their woody tissues probably responded in a similar way as highly susceptible Asian pear wood tissues (Santander et al., 2022b). High susceptibility of rootstock tissues and high canker incidence detected in the current study could explain how E. amylovora concentrations in rootstock samples collected in the current study declined in fall 2017 below the limit of detection of PCR used (Taylor et al., 2001). PCR can have a detection limit of E. amylovora in plant DNA from apple shoot extracts of 10⁵ – 10⁶ CFU/ml with A/B primers (Bereswill et al., 1992) or 3.7 × 10⁵ CFU/ml with RS24580-205 primers (Ham et al., 2022). The G1/G2 primers used in the current study (Taylor et al., 2001) and FER1-F/rgER2R primers (Obradović et al., 2007) have a detection limit of E. amylovora in plant DNA of 10³ – 10⁵ CFU/ml. The PEANT1/2 primers (Stöger et al., 2006) have a detection limit of 10⁴ – 10⁶ CFU/ml (Ham et al., 2022). However, in contrast to shoot extracts, suberized and lignified tissues of bark and xylem from apple rootstocks were used in the current study. Such tissues could significantly impact E. amylovora DNA extraction efficiency and raise the limit of detection of PCR, i.e. lower its sensitivity (Santander et al., 2019; Santander et al., 2022a). Thus, high fire blight susceptibility of M.9 and M.26 rootstocks and sampled lignified tissues could have led to very low E. amylovora concentrations in sampled rootstocks in fall 2017 and no pathogen detection with PCR used in this study.

One year after the 2016 epidemic, in fall 2017, 7.1 – 47.2% of the PCR positive rootstock trees in fall 2016 and dead or cankered central trees recorded in fall 2017, were not removed from the orchards. This demonstrated that on average 22% of rootstock infected trees remained in the orchards, developed cankers, and continued to decline in the year after epidemic. This made it difficult for growers to associate this tree collapse with continued fire blight activity because of the long time for symptom expression on rootstock after the trees were freed of crown symptoms by pruning. The infection foci selected were hypothesized as the most difficult cases to deal with in epidemiological and tree removal respects, because they were largely positioned at the rim(s) of the large groups of fire blight dead trees, i.e., just outside the border of these groups. The focus of the current study was on these foci to determine whether the extent of tree removal needs to be larger in surface area, i.e., expanded beyond the edge of the large groups of dead trees first killed in the epidemic. With 27% latent infections detected in fall 2016, 35% cankered rootstocks and 13.5% dead trees in fall 2017, along with new tree deaths in fall 2018, the area of tree removal should be expanded to just outside of the border of large groups of dead trees. This expanded zone should include the edge trees around the rootstock cankered trees in the infection foci that were examined in the current study. Such practice could prevent continuing tree decline and reduce the need for multiple cycles of tree removal.

The biochemical profiles of the E. amylovora isolates from rootstocks and other plant organs from apple tree orchards in NY state coincided with those described for E. amylovora (EPPO, 2022). The API 20E results were, in general, homogeneous, with slight variability in the metabolism of inositol, gelatin hydrolysis and arginine dihydrolase. These results are similar to those described by other authors (Donat et al., 2007; Végh et al., 2017). The isolates analyzed in the current work also showed diverse EPS production and virulence. Despite E. amylovora’s genome homogeneity (Mann et al., 2013), variability in the virulence and EPS production among E. amylovora strains is usually reported (Puławska and Sobiczewski, 2012; Popović et al., 2020; Mendes et al., 2022). A link between EPS and virulence has also been established (Maes et al., 2001). The most of the phenotypic diversity observed among strains is probably due to different plasmid and genomic island content (Mann et al., 2013).

Although copper has been used as a selective and differential compound for the isolation of E. amylovora on different media (Bereswill et al., 1998; Ordax et al., 2012), and reported as beneficial for the recovery of stressed cells when amended to King’s B medium (Ordax et al., 2012), less than half of the strains analyzed showed improved growth in the presence of copper. The different results in the current study compared to those of Ordax et al. (2012) might be due to the use of a reduced number of strains to characterize the medium, including the European type strain CFBP 1430, which was one of the strains showing growth enhancement in the presence of copper, in the assays of the current study. Additionally, in the current study, two of the analyzed strains showed high susceptibility to copper and were unable to grow on KB amended with the recommended copper concentration. Hence, although the use of copper-amended media may still be useful for the easy differentiation of E. amylovora colonies during isolations, the use of other selective/differential media in parallel is recommended to improve the success of isolation.

This study is the first work that demonstrates high presence (up to 45%) of E. amylovora latent infections in the edge tree rootstocks during the fall season of the epidemic year. The latent infections expressed as rootstock cankers in the following growing season, i.e. with delay, and the incidence of cankers on the edge tree rootstocks (up to 56%) was higher than the percent of latent infections detected by PCR in an epidemic year. As these cankers expressed with significant delay, PCR detections were negative indicating on the likely decline of E. amylovora concentrations in rootstocks to the levels below the PCR detection limit. Up to 35% of the rootstock-cankered edge trees died with a few new tree deaths two years after the first PCR detection. The data of the current study indicates that tree removal due to latent E. amylovora infections of rootstock should be expanded beyond the borders of the dead trees killed early in the epidemic. In conclusion, the recommended removal of trees with latent E. amylovora infections based on the current study would ensure a more complete elimination of E. amylovora-infected apple trees in the orchards and help improve disease management and replanting efforts.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

SGA originated the project ideas and questions in consultation with apple growers, designed the field experiments, wrote project proposals, and secured funding, provided the necessary laboratory equipment and materials, supervised, and coordinated the work, mentored ŽMP, RDS and CLM, created rootstock graphs and tables, and drafted, reviewed, and edited the manuscript. RDS optimized processing of rootstock samples for PCR, optimized PCR extraction conditions, co-mentored ŽMP, conducted all the assays for characterization of E. amylovora strains, and co-drafted, reviewed, and edited the manuscript. CLM performed infection foci selection, field tree and rootstock labelling, and rootstock sampling, rated canker symptoms and tree health status, recorded data in paper and electronic files, and reviewed and edited the manuscript. ŽMP processed rootstock samples, performed DNA extractions, contributed to the optimization of PCR conditions, conducted PCR detection assays, and reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.