A completely randomized full-factorial design was used to organize the individuals into four treatment groups: droughted and inoculated with N. australe , droughted and not inoculated , watered and inoculated with N. australe and a control; watered and not inoculated . Data were collected for ~90 days to track declines in health and mortality rates among the different treatments. Drought-treated plants received 1 L of water on the day of inoculation and another 0.5 L on day 38. Those with no drought treatment received 0.5 to 1.0 L of water by hand once per week depending on soil moisture, which was monitored regularly using a TDR machine from Soil Moisture Co. . Soil moisture for non-drought plants was maintained between 15–25% moisture for the entire experiment. Cultures for inoculations were made from re-isolations of field samples that were collected in January 2016 and positively identified to be N. australe . Inoculations took place on 3 November 2016 , nft hydroponic using methods adapted from Michailides and Swieckiand Bernhardt . Mycelial plugs were made from 8-d-old cultures growing on halfstrength potato dextrose agar amended with streptomycin to prevent bacterial contamination. Plants were first sprayed with 70% isopropyl alcohol to sterilize the surfaces and surrounding areas.

Mycelial plugs were taken from the advancing margin of N. australe cultures and placed on strips of Parafilm using sterile petroleum jelly for adhesion. Plugs were then placed to superficial wounds made on the main stem . The Parafilm strips were then gently wrapped 2–3 times around the stem to keep the plugs in place and prevent contamination. Those plants not receiving fungal inoculation received a control inoculation with uncultured potato dextrose agar using the same techniques. 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. .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, 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 .

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. The dates each plant was harvested and structural data at harvest were recorded. 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, 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.Discoloration of leaves occurred on individuals experiencing both drought- and fungal-related stress . Leaves of drought-treated plants turned from green to brown as drought stress progressed, and leaves from inoculated plants turned brown or dark grey/black. No fungal symptoms appeared in the D– group throughout the experiment; therefore, all stress symptoms in this group were presumed to be drought-related. There was a significant interaction effect between watering regime and inoculation treatment on stress severity in Week 10 . Stress symptoms appeared most rapidly in the D+ and W+ treatment groups, and by Week 5 both of these groups had greater stress symptom severity than the control plants. The D+ treatment group had greater mean stress severity than the D– group but was not significantly different from the W+ group. By Week 10, the interaction between watering and inoculation had strengthened and although all individuals in the D+ treatment group had the highest levels of disease severity , all treatment groups exhibited higher mean disease index scores than the control group .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.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, hydroponic gutter 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 , 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.