Optimum values for Fv/Fm are 0.80–0.83 in C3 plants . Mortality of an individual was assessed by using the stress index, inspecting the texture of the leaves, and evaluating gas exchange and dark-adapted fluorescence values. An individual was determined dead and harvested immediately if it had a stress index score of five or higher, leaves were crispy instead of flexible, and if at least two leaves measured Anet and Fv/Fm values of less than 0.5 µmol CO2×m-2 ×s-1 and less than 0.300, respectively. Stems of harvested plants were checked for lesions by scraping the bark away from the POI and looking for darkened, necrotic tissue extending upward from the POI. Lesion length was measured in centimeters from the POI to the farthest advancing margin of the lesion. Soil moisture, plant structure, physiological data, and disease severity were statistically compared using ANOVA in JMP, version 14 Pro , and post hoc analyses of means were performed using Wilcoxon signed rank test. Two-way factorial ANOVAs were conducted on the influence of watering regime , inoculation treatment , and interaction effects between watering regime and inoculation treatment on plant Anet, Fv/Fm, and disease severity. Correlations between disease severity and physiological stress responses were also examined in JMP using a linear regression analysis to determine maximum fit. Survivorship of each treatment group was estimated using the Kaplan–Meier survival analysis with the survival package in R v. 3.5.1 .
A Cox proportional-hazards model was followed by a Peto and Peto post hoc test to test for statistical significance of Kaplan–Meier survivorship. Due to the small sample size of individuals available for the experiment, procona buckets all reported results for survival were based on a 90% confidence level, and P-values above 0.05 but below 0.1 were considered significant trends. All other tests were conducted using a 95% confidence level for significance.Both chlorophyll fluorescence and net photosynthesis declined as hosts were exposed to drought and fungal infection. Each of these factors caused measurable physiological stress in A. glauca individually; however, in combination, stress symptoms showed up earlier and more strongly . Additionally, an important result was the relationship between visible stress symptom severity and physiological function. Both Anet and Fv/Fm were found to be highly correlated with visible signs of stress that ultimately led to plant mortality. This is consistent with previous studies that have found that Fv/Fm correlates strongly with eventual mortality, and therefore, can be an indicator of drought-related mortality risk in natural systems . Furthermore, Anet was shown to decline even with very low levels of visible stress, suggesting it may be valuable as an early detector of plant stress even before major visible symptoms appear. While Anet and Fv/Fm can be useful tools for measuring physiological stress, they are expensive and difficult to measure on the ground at large scales. Therefore, using visible stress severity indices may be a promising and cost-effective method with which to quickly carry out large surveys aimed at predicting drought- and fungus-related mortality in the field. High mortality was observed in all inoculated plants regardless of drought treatment, indicating that N. australe may act as an obligate pathogen on A. glauca, at least in young, small individuals as were used in this study.
However, mortality occurred much faster in the D+ group. Additionally, some individuals in the D- and W+ group at Week 10 survived well beyond the termination of experiment , suggesting the ability of A. glauca to allocate sufficient resources for defense against drought stress, and in some cases, infection by N. australe, but a greater vulnerability in the simultaneous presence of both factors. Therefore, it appears that a synergistic interaction does exist whereby exposure to both drought and infection by N. australe yields more accelerated decline than either factor alone. It is likely that A. glauca susceptibility, or “predisposition” to disease , is due to the interactive roles of water and carbon availability in plant defenses against drought stress and biotic invaders, as modeled by McDowell et al. and Oliva et al. . Their framework describes a system in which plant hosts are able to allocate resources to either survive extreme environmental stress or defend against biotic invasion, but may succumb via depleted carbon resources when exposed to multiple stressors. For example, hosts like A. glauca can persist through drought with high resistance to cavitation . They can also divert carbon resources to block the spread of pathogens . However, the combination of global-change-type drought and infection by pathogens like N. australe may leave these hosts vulnerable when they no longer possess the resources needed to simultaneously resist cavitation and invasion by the pathogen.
Furthermore, extreme drought conditions can enhance optimal conditions for the growth of pathogens like Botryosphaeriaceae fungi that thrive in more negative water potentials than the host can withstand . These factors combined can push the host beyond a threshold, increasing branch dieback and ultimately increasing the likelihood of whole plant mortality. Understanding the role of pathogens and drought stress in native vegetation canopy loss has long been of great interest to ecologists, though research involving such systems has yielded varying results regarding these interactions. For example, a meta-analysis by Jactel et al. found that in studies on the effects of pathogens and insects on forest plant hosts during drought, damage to hosts varied greatly based on the feeding habits and substrate of the pathogen and severity of the water stress. In the case of secondary agents , more damage occurred on hosts experiencing water stress compared to non-stressed controls, and damage severity increased with increasing water stress. These findings are consistent with the results of the present study and support the hypothesis that drought stress predisposes hosts to pathogen impacts. Other studies have found similar results regarding secondary pathogens in drought-tolerant plant systems, including red pine forests , eucalyptus forests , and chaparral shrublands . By contrast, Davis et al. concluded that drought-induced cavitation alone, not infection by Bot. pathogens, caused canopy dieback of southern California Ceanothus sp. during drought, suggesting that secondary agents do not always benefit from drought-related predisposition. Clearly, while secondary pathogens are known to become pathogenic in hosts experiencing environmental stress , the mechanisms driving this relationship in different plant hosts are not fully understood. Although field studies have been conducted on the presence of fungal pathogens on shrubland species during drought , controlled experiments manipulating both drought and fungal treatments in naturally occurring species are rare and typically involve tree systems rather than wildland shrub species . To the authors’ knowledge, this is the first experiment to investigate the influences of drought and infection by N. australe on A. glauca by manipulating both factors.The results of this experiment, along with the identification of N. australe and other Bot. species in the region , suggest that the severe canopy dieback of A. glauca observed in Santa Barbara County between 2012 and 2016 is likely the result of global-change-type drought combined with the presence of opportunistic fungal pathogens like N. australe. While there is evidence to suggest that acute drought alone may cause some mortality in A. glauca , procona florida container the presence of N. australe and other pathogens likely exacerbates stress and accelerates mortality in these hosts. Furthermore, N. australe has long been reported in avocado orchards in Santa Barbara County ; however, there are no known reports or indications of major disease and dieback of A. glauca in surrounding chaparral shrubland system until recently, during the especially dry winters of 2013 and 2014 .
Thus, we suspect that while N. australe has likely been present on A. glauca hosts , the drought of 2011–2018 was the most severe in the region in the past 1200 years and may have been significant enough to push adult A. glauca past a tipping point of defensibility against N. australe. It should be noted that results of experiments on young plants, which may be highly susceptible to drought and drought-related mortality due to limited carbon reserves, may not scale directly to large, mature individuals in the field . This study showed high mortality in 2-year-old A. glauca exposed to a fungal pathogen with and without drought, in contrast with field observations of diseased, large adults exhibit severe canopy dieback and are ridden with fungal cankers, yet still survive . Previous studies have yielded similar results: for instance, photosynthesis was shown to be greatly reduced in oak seedlings compared to adults in drought years compared to wet years , and He et al. reported that responses of red maple and paper birch saplings to a 1995 drought were significantly different than those of mature adults. Similarly, since hosts are often able to allocate carbon reserves to compartmentalize canker-causing agents like N. australe within carbon-rich barriers , larger individuals with more biomass and greater carbon stores are able to utilize and direct more resources to defense than younger, smaller individuals. Thus, mature plants can better persist through biotic attack during environmental stress than their younger counterparts and experience various levels of canopy dieback rather than full mortality. Arctostaphylos glauca are obligate seeders, meaning they are killed by fire and must maintain populations by individuals recruiting from seed rather than resprouting from their base. Therefore, young, small individuals may be of greatest concern for future populations of this species. Because current research is predicting more frequent and extreme drought events , more exotic pathogens , and more frequent fire in these southern California shrublands , populations of A. glauca could decline because small individuals may be highly susceptible to disease and mortality. A valuable next step for understanding these risks and predicting future shifts in vulnerable chaparral communities would be to monitor young recruiting populations of A. glauca for N. australe for signs of stress, infection, and mortality in the wild.In the face of rapid climate change, it is increasingly important to understand the abiotic and biotic mechanisms driving ecological landscape change. Large plant dieback events can produce major ecological consequences, including changes in vegetation cover , increased fire risk , and changes in hydrology , all of which affect ecosystem structure and functioning . Furthermore, the loss of even a few species can trigger effects on the local food web structure , and increase risk of invasion . The results of this study suggest that small individuals of A. glauca, one of the most common and widespread species the southern California chaparral community, are at high risk of disease and dieback due to opportunistic pathogens and extreme drought. The potential for dieback of Arctostaphylos spp., which provide food for animals such as mice, rabbits, and coyotes and are an important component of post-fire woody regeneration in chaparral, raises concerns regarding changes to ecosystem structure and functioning in the coming decades. Many ecosystems today are facing unprecedented drought ; yet, the interactions of drought and pathogens in wild land settings are difficult to study because the multitude of confounding variables and the challenges of manipulating both the pathogens themselves and climate. Thus, greenhouse studies such as this one are increasingly essential to understand the influences of drought and pathogens as they relate to dieback events, as well as to understand the relationship between stress and shrub/tree ontogeny . Critical questions remain regarding the relative tipping points for large-scale dieback among historically drought-tolerant species such as A. glauca that today are facing the combination of extreme drought and novel pathogens. These pathogens may not express themselves until there is drought, highlighting the need for broader field surveys and long-term monitoring of wild land ecosystems. An important step to understanding the role of disease in contributing to vegetation change is also to isolate pathogens and test their pathogenicity under varying controlled conditions. This study provides one such step for what appears to now be a widespread, opportunistic introduced pathogen in an important native California chaparral shrub. Extreme drought events from climate change have produced immediate and dramatic effects in recent years, with costs often exceeding $1 billion due to their widespread economic and ecological impacts . Among the ecological consequences is widespread tree mortality, , event within plant systems that have historically been considered drought-tolerant .