To confirm Koch’s postulates, the standard criteria to determine the agent causing a disease , we reisolated fungi from stem tissue at least 2-cm above the point of inoculation in harvested plants, amplified using primer pairs ITS1F/ITS4 for the ITS and EF1-728F/986R for alpha-elongation factor-1 . They were sequenced using the protocol described by Schultheis et al.. Daily temperatures were maintained between 18–30°C during the day and 10–15°C at night. Humidity maintained at 50%. Photosynthetically active radiation lights were set to provide 14 h of daylight per day and a maximum of 2000 µmol. Plant positions were randomized weekly using a random number sequence generator to eliminate any microclimate effects in the greenhouse.Physiological stress due to drought and pathogen infection was inferred from weekly measurements of net photosynthesis and dark-adapted chlorophyll fluorescence using a LI-COR 6400XT and Hansatech FMS2 system fluorometer , respectively. Leaves were dark-adapted using leaf clips for 20–30 min before measuring fluorescence. One healthy , fully expanded leaf per plant, round plastic pots or on one healthy and one stressed/diseased leaf per plant if symptom onset had begun . All data were collected between 10:00 hours and 16:00 hours to capture peak values for the day, with the majority of measurements taken between 10:00 hours and 12:00 hours.
Due to mechanical issues, chlorophyll fluorescence was not measured on 4 and 11 November and on 20 December 2016. Net photosynthesis and dark-adapted fluorescence were chosen as proxies for plant health, as lower values correlate strongly with higher levels of drought stress .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, 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.The results of this study support the hypothesis that drought stress reduces resistance to pathogens in A. glauca, and fungal infection enhances plant mortality compared to drought alone.
As predicted, both physiological metrics showed declines as drought and disease progressed, suggesting rapid plant responses to both stresses. Furthermore, a strong correlation was found between declining physiological function and increases in stress severity index, suggesting that visible signs of stress may be used to assess physiological decline and reveal more severe underlying problems in the field. Finally,although mortality rates for inoculated groups were similar, drought-stressed A. glauca shrubs infected with N. australe trended toward faster and greater mortality than in any other treatment group.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 , 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 Naustrale 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 thanresprouting 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, hydroponic bucket 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 wildland 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 . While seasonal droughts are known to be a natural and regular occurrence in arid and semi-arid regions, the increased frequency, duration, and intensity with which they have occurred in recent years is highly unusual . Such extreme droughts, referred to as “global-change type drought” , are predicted to continue, and even become the norm, as a result of human-induced climate change . Consequently, species that are typically capable of withstanding regular drought stress may be susceptible to canopy dieback, and mortality, as a result of shifts in drought regimes . One such plant community that may be vulnerable to extreme climatic change is chaparral. Chaparral shrublands, which occupy approximately 7 million acres throughout California , are a dominant vegetation community in southern California, composed primarily of evergreen, drought tolerant shrubs and subshrub species including manzanita , ceanothus , and chamise . These species are well adapted to the seasonal variations in temperature and precipitation typical of mediterranean climates where hot, rainless summers are the norm . However, mediterranean-type regions like southern California are predicted to experience rapid increases in temperature , and increased drought occurrence and severity ; IPCC, 2013, resulting from human-caused climate change. These regions have thus been designated as worldwide global change “hot spots” . Indeed, recent studies have reported extensive mortality of chaparral shrub species resulting from global-change type drought throughout southern California . Thus, climate change represents a significant threat to native plant community persistence in this region. A critical topic for ecological research is understanding where, how, and to what extent plant communities will change as a result of increased drought . Studies aimed at understanding the physiological mechanisms behind drought-related plant mortality – and why some plants suffer mortality from drought while others survive – have elucidated a variety of complex mechanisms of plant mortality . These include loss of hydraulic conductance , exhausted carbon reserves , and susceptibility to pests and pathogens due to being in a weakened state from drought . Measuring xylem pressure potential can be a useful index of soil water availability , and dark-adapted fluorescence can be a quick and accurate indicator of plant stress, as values drop significantly in water-stressed plants,. Together, these may be useful tools for predicting plant vulnerabilities to drought and biotic invasion. Landscape variables such as elevation, slope, and aspect have also been shown to correlate with plant water stress and mortality , and can be useful for predicting vulnerabilities during drought. However, major knowledge gaps still remain, and studies combining field mortality patterns with physiological data on plant water stress are rare . Plants employ a variety of complex strategies to cope with drought stress, but generally fall along a continuum of “drought avoiders” or “drought tolerators”. Drought avoidance, also known as “isohydry”, refers to plants that regulate stomatal conductance to maintain high minimum water potentials as soil dries out . While this strategy reduces the risk of xylem cavitation and subsequent hydraulic failure, it may increase the likelihood of carbon starvation, as C assimilation is greatly reduced . Conversely, drought tolerant plants maintain higher Gs, even at very low water potentials, which allows for continued C assimilation but with greater risk of xylem cavitation . These different strategies can have significant implications for ecosystem level consequences of severe drought ; indeed, recent studies have linked anisohydry with greater levels of mortality in chaparral systems . An historic drought in southern California provided an opportunity to simultaneously measure physiological stress and dieback severity along an elevational gradient in aclassically drought-tolerant evergreen chaparral shrub, big berry manzanita . A. glauca is one of the largest and most widely-spread members in a genus consisting of nearly 100 species.