Additionally, our study provides valuable insight into areas of greatest risk for dieback and mortality, which are predominantly in lower elevations. These are important factors to consider when predicting vulnerabilities and potential impacts of future extreme drought events . Mediterranean shrublands like those in southern California already considered high risk for global-change type drought, , and research suggests a general trend of upwards-shifting ranges in southern California chaparral species driven by changes in climate . Therefore, populations of A. glauca occurring at the lower edge of their natural range are at high risk for dieback and mortality, and should be the focus of management efforts. Lastly, while studies on the various physiological mechanisms for plant survival during drought are critical for predicting differential responses to stress, there is an increased emphasis on the importance of understanding the diverse role of pathogens in order to accurately model species vulnerabilities to climate change . Studies that incorporate the impact of pathogens help inform new integrative approaches to protecting plants against drought and biotic infection, rather than treating these influences separately. Examples include Jactel et al., , whose meta-analysis showed the significant effects of water stress on symptom severity in plants infected with latent pathogens like Bots, and experiments like Drake-Schultheis et al. , procona system who found interactive effects between drought stress and infection from N. australe in driving symptoms of stress and increasing mortality rates in A. glauca.
The results of our study align with these frameworks, and provide additional evidence that as climate change models are predicting more intense and frequent drought events, our need to understand the role of latent pathogens in at-risk natural systems is becoming more critical.Reports of large-scale, drought-associated mortality events in forest and woodland systems have been on the rise in recent decades . These reports have spanned across biomes, including in classically drought-tolerant species across Europe , Australia , Africa , and the United States . As a result, interest has been growing in understanding how species that are typically capable of withstanding periodic drought stress may become susceptible to drought and experience significant dieback and even large-scale mortality when exposed to acute or prolonged chronic drought . These droughts of unusual extremes are referred to as “globalchange-type drought” and are becoming more common as the climate warms . While the exact physiological mechanisms leading to dieback and mortality during such events are variable across species and conditions, drought is generally hypothesized to promote physiological decline either via loss of hydraulic functioning or carbon starvation or a combination of both . In the case of hydraulic failure, plants with insufficient soil water experience xylem cavitation , which can ultimately lead to cellular death. Alternatively, plants that avoid drought by closing their stomata to reduce water loss subsequently suffer insufficient carbon supply to meet other metabolic demands. In either scenario, the stress that drought places on a plant is likely to cause measurable decreases in physiological functions that may be irreversible .
An additional factor that can play a significant role in drought-related dieback and mortality is the presence or introduction of biotic agents. Indeed, introduced plant pathogens have been well documented to cause canopy dieback and dramatically alter community structure in a variety of forested systems . Some well-known examples in the United States include Dutch elm disease , chestnut blight , white pine blister rust , and sudden oak death . Significant pathogen events have also impacted the landscape in wild land shrub communities including sclerophyll shrub woodlands in Australia and salt desert scrub in the western United States . However, large-scale dieback of shrubs has been less documented than their arboreal counterparts, despite evidence of disease from fungal species being abundant in many scrubland systems including southern California chaparral , northern California foothill shrublands , and South African fynbos . Such studies, along with expectations of increasing threats from pathogens due to climate change and accelerating trade/movement of biological materials globally , have led scientists and land managers alike to anticipate introduced pathogens as important contributors to future changes in wild land communities.While both global-change-type drought and pathogens are likely important contributors to plant dieback and mortality, current research suggests that these two factors are not mutually exclusive . Rather, canopy dieback and mortality may result from the combined influences of environmental stress and biotic agents, and theoretical frameworks describing these influences have been put forth .
These frameworks incorporate biotic agents into the drought-hydraulics complex described above, whereby pathogens and insects may amplify or be amplified by drought-associated hydraulic failure or carbon starvation . Amplification can occur when biotic agents damage host tissue—by defoliation or blocking transportive vessels, for example—to the extent that the effects of drought are greatly exacerbated . Alternatively, physiological responses to extreme environmental stress can have negative effects on plant defense systems, rendering them susceptible to mortality through biotic infection . In both scenarios, the effects of biotic agents and drought stress are strongly linked, and these interactions have been well documented in drought-tolerant systems such as South African fynbos , red pine forests , eucalyptus forests , and California chaparral . Latent or secondary pathogens are particularly likely to be involved with dieback and mortality events in these systems, as they are known to increase damage in hosts experiencing drought stress . Therefore, while drought events alone are expected to play an important role in reshaping ecosystems as the climate changes, in some cases, synergies between environmental stress and biotic influences might lead to shifts in plant community structure and composition, and thus ecosystems as a whole. In the Santa Ynez Mountains in Santa Barbara County, California, United States, big berry manzanita began exhibiting dramatic canopy dieback during the 2011–2018 drought . Shrubs in the genus Arctostaphylos are common in Mediterranean shrub communities extending from southwest Oregon to northern Baja California . They may occur in monospecific stands or in alliances with other important community members like chamise and Ceanothus spp. . Within these alliances, Arctostaphylos spp. frequently occupy >50% average cover , which along with their nutritious and prolific fruits, and fire-induced regeneration strategies, make them one of the most important members of the chaparral community . In the southern California chaparral ecosystem where hot, dry summers with high vapor pressure deficit are the norm , seasonal drought tolerance has long been considered a common strategy among dominant plant species, including A. glauca. However, the severity of recent canopy dieback observed suggests that this species is reaching a threshold in its drought-resistance capability. Concurrent with observations of canopy dieback, visible symptoms of fungal infection were observed including wood cankers and leaf discoloration , both ofwhich progress during prolonged drought stress, suggesting that multiple driving forces contribute to manzanita dieback. Molecular sequencing identified the dominant fungal pathogen found on symptomatic A. glauca in this area to be Neofusicoccum australe, a member of the well-known pathogenic Botryosphaeriaceae family . Members of this family are most commonly associated with disease in plant species experiencing severe environmental stress , including Arctostaphylos spp. . They are also known to play a variety of functionally diverse roles, from asymptomatic endophytes to obligate pathogens . Yet, while N. australe has been described around the world , relatively few studies have been conducted on its specific interactions with host species, procona valencia buckets as it was only fairly recently described . Historically, Bot. pathogens have most frequently been studied in agricultural host species , and little is known regarding their ecological role in wildland ecosystems , especially with regards to chaparral shrubland systems . The present study was aimed at identifying the possible role of N. australe in A. glauca dieback in Santa Barbara County, particularly in combination with extreme drought. Because this pathogen has only recently been reported on wild shrub species in California and is thought to be an introduced species native to Western Australia , this outbreak represents a new and undescribed threat to these wild land plant assemblages. This study addresses the following questions: How does A. glauca respond physiologically to drought and fungal infection, separately and together?
Are these responses correlated with visual signs of stress, specifically leaf health? Can drought and fungal presence interact to increase or accelerate plant mortality compared to drought or fungi alone in A. glauca? To address these questions, a greenhouse experiment was conducted in November 2016 through February 2017 manipulating both drought and fungal infection and observing trends in plant stress symptoms, physiological function, and mortality. We predicted that both drought stress and fungal infection would lead to declines in physiological function compared to the control and that these declines would be strongly correlated with increases in stress severity. Furthermore, we expected that those individuals experiencing both drought stress and fungal infection would die sooner than those in all other treatment groups. This experimental study elucidated the potential of the interaction between drought stress and introduced pathogens to significantly impact chaparral shrub health and important implications for the future of these shrubs faced with increasingly frequent globalchange-type droughts. 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 , using methods adapted from Michailides and Swiecki and Bernhardt . Mycelial plugs were made from 8-d-old cultures growing on half strength 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. . Greenhouse conditions were set to reflect summer conditions in Santa Barbara. 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, 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 .