Evergreen Oak Decline Associated with Invasive Phytophthora spp.: Structure and Functionality of Soilborne Microbial Communities in Response of Application of Biofumigation
Author(s)
Aurangzeb, Wajid
Date Issued
May 9, 2025
Type
Doctoral Thesis
Abstract
Chapter 1 provides a comprehensive background to the study, emphasizing the significance of cork and holm oak species and their ecological and economic importance. It explores the history of oak dieback, focusing on the role of Phytophthora species in the decline of these trees. Additionally, the chapter highlights various tools for identifying, detecting, and characterizing Phytophthora species, with a particular emphasis on molecular methods such as metabarcoding, which forms a core methodology in this thesis. It also discusses the available control methods, both chemical and biological, that are vital for integrated disease management strategies against Phytophthora.
Chapter 2 focuses on a series of experiments conducted to explore the diversity of Phytophthora species in the Castelporziano Presidential Estate, central Italy. The primary goal was to combine traditional detection methods with modern molecular identification using high-throughput sequencing (HTS). While P. cinnamomi role in ink disease is well established, research is ongoing into the roles of other soil-borne Phytophthora species. In this context the pathogenicity trials of P. multivora, (recently isolated Phytophthora spp. in Italy) was
conducted on on cork and holm oak seedlings in mesocosm. The results showed that this species can cause significant damage to both holm and cork oaks. Phytophthora multivora is a fine root feeder, and in Australia, it has caused damage to native plants. Its ability to thrive in dry environments can make it a more serious threat to cork and holm oaks, especially looking at the climate change projections. The HTS experiments, combined with baiting, confirmed the presence of P. cinnamomi, P. multivora, and other Phytophthora species at the estate. In total, 52 rhizosphere soil samples were collected from declining oak stands, leading to the isolation of 254 oomycete taxa, including P. cinnamomi, P. multivora, P. plurivora, P. gonapodyides, and P. bilorbang. HTS revealed 20 different Phytophthora species, with P. cinnamomi being the most prevalent. Interestingly, we found no significant difference in the Phytophthora community between tree species.
Chapter 3 focused on the evaluation of a biocontrol strategy aimed at combating cork oak ink disease caused by P. cinnamomi. The main goal was to determine the optimal dosage of a BioFence FL® product that could effectively reduce the P. cinnamomi inoculum under field conditions. BioFence®, a commercially available seed extract of Brassica carinata, in two forms: pellets (BioFence®) and a liquid (BioFence FL®) were tested. While BioFence® is known to work against soil-borne diseases, the right dosage and product form were unclear for BioFence FL ®. A comparative In vitro study was conducted to evaluate the potential of both products against P. cinnamomi inoculum at soil depths of 5 and 15 cm. The results showed that BioFence FL® was effective at both depths resulting in total inhibition of P. cinnamomi. Based on results, further In vitro and field trials were conducted to determine the minimum effective dosage. A 2% concentration of BioFence FL® was found to successfully kill P. cinnamomi inoculum in both lab and field condition. Additionally, preliminary analysis of the
impact of BioFence FL® was observed on microbial communities (fungi and bacteria) using culture-based method and results showed an increase in bacterial colony-forming units (CFU) while reducing the number of fungal CFU. Based on these results further studies were conducted to understand the impact of biofumigation on microbial community which is the focus of next chapter.
In Chapter 4, the impact of biofumigation on the soil microbial community was observed, focusing on bacteria, fungi, and oomycetes, using HTS to assess the changes in microbial composition and diversity. Two experiments were carried out simultaneously. In the first experiment, DNA extraction was performed directly from biofumigated soil to analyze the microbial community. In the second experiment, soil was cultured on growth media, extracted DNA from the resulting colonies, and then subjected those samples to HTS analysis. This second approach was particularly novel, making it the first time a culture-based method had been used in combination with HTS to study the effects of biofumigation on microbial communities. Both approaches showed similar results—biofumigation boosted bacterial communities and suppressed fungal and oomycetes populations. Specifically, P. cinnamomi decreased from 9.11% before treatment to 0.25% after treatment in terms of relative abundance. Among fungi, the genus Linnemannia decreased while Mortierella increased in relative abundance. Both genera are considered as ectomycorrhizal fungi. Fungial genus such as Fusarium and Verticillium which are considered as important soil borne fungal genera were also significantly inhibited by the biofumigation. Furthermore, biofumigation had a positive effect on bacterial CFUs, while fungal CFUs declined, reflecting similar observation to direct HTS results. . No oomycete colonies were observed before or after biofumigation in culture-based methods.
Chapter 5 focus on the differences in the soil microbial community present in the rhizosphere of healthy and declining cork oak trees affected by ink disease in Monte San Biagio, central Italy. This forest is home to one of the largest pure cork oak stands in Italy and has been experiencing symptoms of decline since 2001, when P. cinnamomi and other Phytophthora species were first detected. Rhizosphere soil samples from both healthy and declining cork oak trees were collected and subjected to HTS analysis. Although, there was no significant difference in overall microbial diversity between healthy and declining trees (as shown by alpha and beta diversity indices), specific fungal families were observed in either healthy or declining trees. For example, Amanitaceae, Cortinariaceae, and Clavuliniaceae were more abundant in declining trees, while Atheliaceae, Gloniaceae and Heliotales were more prevalent in healthy trees. These fungal families, especially Amanitaceae, Cortinariaceae are ectomycorrhizal fungi that are beneficial for trees by improving nutrient uptake and offering protection against pathogens. Bacterial genera such as CL500-3 marine group and Dongia were more common in healthy trees, whereas Arenimonas and Dokdonella were more prevalent in declining trees. For oomycetes, Pythium was the most abundant genus, followed by several Phytophthora species, with P. cinnamomi being the most dominant. P. cinnamomi was present in both healthy and declining trees. The current findings are crucial towards mitigation strategies against Phytophthora infested forest indicating a need for management practices towards healthy forest sites along with declining trees.
Chapter 6 focuses on the survey of cork oak forests in Monte San Biagio, Central Italy to identify declining cork oak trees, collect soil samples for Phytophthora isolation, and assess the effectiveness of endotherapy (trunk injections) using potassium phosphonate (Kalex®). The survey, conducted in 2020/2021, included soil sampling from 78 cork oak trees to isolate
Phytophthora species. Three different hotspots were selected for treatment and 419 trees in hotspot 1 and 1385 trees in hotspot 2 and 435 trees in hotspot 3 were treated with Kalex® by endotherapy. Disease severity was monitored before and after treatment in hotspot 1 and 3. The survey identified typical symptoms of cork oak decline such as defoliation, yellowing of leaves, shoot dieback, and crown thinning. Soil samples revealed the presence of P. cinnamomi, P. plurivora, and P. cryptogea, with P. cinnamomi being the most prevalent species. In hotspot 1, disease severity decreased from 25.18% before treatment to 22.02% one year later but increased again to 30.21% two years after treatment. Trees with mild to moderate symptoms (disease severity scales 0-2) responded better to the treatment, while those with more severe symptoms (scale 3-4) showed little improvement. In hotspot 3, disease severity was initially 26.09% and decreased to 24.07% one-year post-treatment. These results suggest that potassium phosphonate treatment can be effective for trees in the early stages of decline but is less effective in trees with advanced symptoms.
Keywords: Oak decline, Phytophthora. Biocontrol, Metabarcoding, High-throughput Sequencing (HTS), Rhizosphere, Soil Microbial Community, Disease Management, Biofumigation
Additional information
Dottorato di ricerca in Scienze, Tecnologie e Biotecnologie per la Sostenibilità