The role of yellow lupin (Lupinus luteus) in the decline affecting oak agroforestry ecosystems

Phytophthora cinnamomi is a soilborne pathogen causing root rot in Mediterranean Quercus species growing in ‘dehesa’ rangeland ecosystems. Recently, it has been reported causing wilting and death of Lupinus luteus (yellow lupin), a spontaneous plant in southern Spain rangelands, but also frequently sowed for livestock grazing. In soils artificially infested with P. cinnamomi chlamydospores and planted with different cultivars of yellow lupin, a significant increase in the density of propagules was detected in comparison with the initial levels of inoculum and with the infested but not planted soil (control). In oak‐rangelands in which yellow lupine was planted, isolation and counting of colonies of P. cinnamomi from soil samples have shown the ability of this plant to maintain or even increase the inoculum density and thus facilitate the infection of trees. Results suggested that cultivation of yellow lupin in oak‐rangeland ecosystems should be avoided whether oak trees are affected by root disease caused by P. cinnamomi or not. This leguminous plant can act as an inoculum reservoir or even enhance inoculum soil levels available for oak root infections, exacerbating the oak decline severity in the region.     

Fine root dynamics of oak saplings in response to Phytophthora cinnamomi infection under different temperatures and durations

The belowground effects of Phytophthora cinnamomi on 1‐year‐old saplings of two common oak species in mid‐Atlantic US forests, white (Quercus alba) and red oak (Q. rubra), were examined after incubation in pathogen‐infested soilless potting mix. Fine root lengths (0–1.5 mm in diameter) of both oak species were quantified after incubation at successive 30‐day intervals up to 300 days, for a total of 10 incubation periods. In addition, colony‐forming units (CFU) of P. cinnamomi were quantified after white oak saplings were incubated in infested soilless potting mix at different temperature/duration combinations that reflect soil conditions present in the mid‐Atlantic United States. Impact of P. cinnamomi on fine root lengths of red and white oak saplings varied considerably over time. Significant periods of fine root loss occurred primarily during spring, when bud break and leaf flush began for both oak species. Red oaks had 17% fine root loss on average, while white oaks appeared more resistant to P. cinnamomi infection with a 2% decrease in fine roots over the course of the experiment. Phytophthora cinnamomi CFU declined significantly with exposure to all incubation temperatures except 8°C. This was in contrast to in vitro experiments, where the optimum temperature for mycelial growth was determined to be 21°C and above. Significant fine root loss caused by P. cinnamomi depended on plant phenology and the oak species tested. Extreme soil temperatures have a significant adverse impact on temporal changes of P. cinnamomi population.     

Viability of holm and cork oak seedlings from acorns sown in soils naturally infected with Phytophthora cinnamomi

The emergence and survival of pregerminated holm oak (Quercus ilex) and cork oak (Quercus suber) acorns from two ecologically different dehesas (Mediterranean open woodlands) were studied in two soils from these stands naturally infected with Phytophthora cinnamomi, and in the same soils previously sterilized in the autoclave. Phytophthora cinnamomi was consistently isolated from the radicles of all unemerged and all emerged but dead seedlings from the unsterilized substrates. Seedlings of holm oak were more susceptible to P. cinnamomi than those of cork oak. Mortality of holm oak seedlings was significantly different depending only on soil treatment (sterilized or unsterilized), and it was 100% in unsterilized soils, independent of acorn provenance and soil origin. Mortality of cork oak seedlings was significantly different depending on the acorn origin and soil treatment, and on the interactions acorn origin × soil origin and soil origin × soil treatment. The demonstrated high susceptibility of holm and cork oak young seedlings to P. cinnamomi could be a limiting factor in Mediterranean open woodlands (dehesas) not only in natural regeneration processes but also when reforestation by direct sowing is implemented.     

Temporal metabolic profiling of the Quercus suber–Phytophthora cinnamomi system by middle‐infrared spectroscopy

The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest ‘hotspots’ for biodiversity in the world. Here, we applied Fourier transform‐infrared (FT‐IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P. cinnamomi mycelium during the early hours of the interaction. As early as 2 h post‐inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock‐inoculated plants. These variations were further extended at 8 hpi. Surprisingly, at 16 hpi, the metabolic changes in inoculated and mock‐inoculated plants were similar, and at 24 hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8 hpi. Principal component analysis of the FT‐IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock‐inoculated plants at 2, 8 and 24 hpi, but not at 16 hpi. FT‐IR spectral analysis from mycelium of P. cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24 h post‐exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24 hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT‐IR technique can be successfully applied to discriminate infected cork oak plants from mock‐inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P. cinnamomi were also altered when exposed to cork oak root exudates.     

Screening brassicaceous plants as biofumigants for management of Phytophthora cinnamomi oak disease

Brassicaceous plants rich in glucosinolates have been used as biofumigants for the management of soilborne pathogens. Efficacy of Brassica plant tissue has mainly been attributed to toxic isothiocyanates released upon the hydrolysis of glucosinolates. Management of Phytophthora cinnamomi, the causal agent of oak root rot in rangeland ecosystems using biofumigation, is promising, but requires further validation. The biofumigation activity of 14 brassicaceous plants was evaluated under experimental conditions. All evaluated plants rich in sinigrin suppressed (100%) the mycelial growth of P. cinnamomi, while plants rich in aromatic or other aliphatic glucosinolates had little or no suppressive effect. Simulating soil amendment in field conditions, the effects on natural soil artificially infested with P. cinnamomi chlamydospores were examined with Brassica juncea, Eruca vesicaria and Lepidium sativum, three species with different glucosinolate profiles. Only B. juncea decreased the viability of chlamydospores significantly in comparison with untreated soil only 1 day after biofumigation, whereas E. vesicaria needed 8 days to reach significance and L. sativum had no effect at all. Despite the decreases in soil inoculum, biofumigation with B. juncea did not prevent the root infections in a highly susceptible host (Lupinus luteus). However, biofumigation with plants rich in sinigrin, such as B. juncea, decreased P. cinnamomi soil inoculum under the experimental minimum threshold for oak disease expression. Although biofumigation should be considered as an effective measure to be incorporated in integrated control of the oak disease, biofumigation by itself would not be effective enough for the substantial suppression of P. cinnamomi inoculum.     

Effects of root damage associated with Phytophthora cinnamomi on water relations,biomass accumulation,mineral nutrition and vulnerability to water deficit of five oak and chestnut species

The effects of root damage associated with Phytophthora cinnamomi on water relations, biomass accumulation, mineral nutrition and vulnerability to water deficit were investigated in pedunculate oak (Quercus robur), red oak (Quercus rubra) and holm oak (Quercus ilex) saplings over two years. Comparison was made with sweet chestnut (Castanea sativa), a susceptible species to infection by P. cinnamomi, and with a resistant hybrid chestnut (Castanea crenata × C. sativa). Trees were inoculated in 1998 and were subjected to water shortage in 1999. All inoculated sweet chestnuts died before the application of water shortage. Hybrid chestnut, pedunculate oak and red oak displayed low root susceptibility to P. cinnamomi. In these species, water relations, aerial growth and mineral nutrition were slightly affected by inoculation. By contrast, holm oak was the most susceptible oak species to P. cinnamomi as inoculated well‐watered trees displayed the highest root loss (67%) and a 10% mortality. Root loss was associated with a decrease in predawn leaf water potential, a 61% reduction in stomatal conductance, a 55% reduction in aerial biomass, a decrease in leaf carbon isotope discrimination and reduced leaf N and P contents in comparison with controls. In hybrid chestnut and pedunculate oak, water shortage resulted in a similar decrease of predawn leaf water potential, stomatal conductance and aerial biomass in inoculated and non‐inoculated trees. In red and holm oaks, soil volumetric water content of inoculated trees subjected to water shortage remained high. The effects observed in those trees were similar to those of inoculated well‐watered trees and were probably the result of root infection only.     

New insights into the survival strategy of the invasive soilborne pathogen Phytophthora cinnamomi in different natural ecosystems in Western Australia

Despite its importance as one of the most notorious, globally distributed, multihost plant pathogens, knowledge on the survival strategy of Phytophthora cinnamomi in seasonally dry climates is limited. Soil and fine roots were collected from the rhizosphere of severely declining or recently dead specimens of 13 woody species at 11 dieback sites and two dieback spots and from healthy specimens of five woody species at four dieback‐free sites in native forests, woodlands and heathlands of the south‐west of Western Australia (WA). Phytophthora cinnamomi was recovered from 80.4, 78.1 and 100% of tested soil, fine root and soil–debris slurry samples at the 11 dieback sites, in some cases even after 18‐month storage under air‐dry conditions, but not from the small dieback spots and the healthy sites. Direct isolations from soil–debris slurry showed that P. cinnamomi colonies exclusively originated from fine roots and root fragments not from free propagules in the soil. Microscopic investigation of P. cinnamomi‐infected fine and small woody roots and root fragments demonstrated in 68.8, 81.3 and 93.8% of samples from nine woody species the presence of thick‐walled oospores, stromata‐like hyphal aggregations and intracellular hyphae encased by lignitubers, respectively, while thin‐walled putative chlamydospores were found in only 21.2% of samples from five woody species. These findings were confirmed by microscopic examination of fine roots from artificially inoculated young trees of 10 woody species. It is suggested that (i) the main function of chlamydospores is the survival in moderately dry conditions between consecutive rain events and (ii) selfed oospores, hyphal aggregations, and encased hyphae and vesicles in infected root tissue of both host and non‐host species are the major long‐term survival propagules of P. cinnamomi during the extremely dry summer conditions in WA.     

Treatment of oak decline using pressurized injection capsules of antifungal materials

Quercus ilex and Quercus suber trees growing at several sites in Extremadura, Western Spain, that were showing symptoms of oak decline were injected with potassium phosphonate, quinosol or carbendazim using a low-pressure method of trunk injection composed of a pressurized capsule system. A team of four people injected between 120 and 189 trees per day, depending upon the density of the undergrowth vegetation. This labour cost represented, approximately, 15–20% of the total cost of the treatment. The potassium phosphonate-injected trees showed a significant improvement in vegetative growth within 2 years of the injection treatment, and they also showed some recovery from the decline symptoms during the third year. Only one injection treatment of an average of 3.5 capsules (corresponding to 24.5 g phosphonic acid) per tree of approximately 36 cm in diameter, was necessary to reduce the disease severity significantly. Indirectly, these results corroborate the implication of Phytophthora cinnamomi in oak decline within Spanish Quercus woodlands.     

Occurrence of Phytophthora cinnamomi in cork oak forests in Italy

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re‐isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.     

Susceptibility to Phytophthora cinnamomi of the commonest morphotypes of Holm oak in southern Spain

The four main morphotypes of Holm oak (Quercus ilex subsp. ballota) present in Andalusia (expansa, macrocarpa, microcarpa and rotundifolia) were infected with Phytophthora cinnamomi to determine their susceptibility to the root pathogen. No large differences were found among the four morphotypes in the infection of roots, which always showed a high degree of necrosis. However, the different responses of the foliage to infection separated the four morphotypes of Holm oak into three groups: very susceptible (microcarpa), susceptible (expansa) and moderately susceptible (rotundifolia and macrocarpa). The natural hybrid Q. ilex ballota–Q. faginea exhibited a low level of root and foliar symptoms when infected with P. cinnamomi. Quercus faginea could be considered as a source of resistance to P. cinnamomi in future breeding programmes.     

The long‐term survival of Phytophthora cinnamomi in mature Banksia grandis killed by the pathogen

The ability of Phytophthora cinnamomi to survive long dry periods is the key to its persistence in the south‐west of Western Australia. It has been proposed that dead Banksia grandis are a significant long‐term reservoir for P. cinnamomi inoculum. To test this, 36 healthy B. grandis trees were inoculated in April 1999, and the presence of viable propagules in planta was determined between 2 and 34 months after tree death. By 10 months after inoculation, 75% of the trees had died, with the remaining seven trees dying by 22 months. The pathogen was more commonly recovered from bark than from wood, except from those trees that died at 22 months, and more commonly from above‐ground trunks than below‐ground trunks and roots until 8 months after plant death. In trees that died 12 months after inoculation, P. cinnamomi was recovered from 60% of trunk and root core samples at 3 months, declining to 33% at 10 months, 5.5% at 12 months and 0.1% at 34 months after tree death. In trees that died at 22 months, P. cinnamomi was recovered from 87% of trunk and root samples 2 months after tree death, decreasing to 0.5% by 33 months. This study suggests that the pathogen does not have a saprotrophic phase within dead B. grandis tissue, and B. grandis is unlikely to be a long‐term reservoir for P. cinnamomi. However, the manipulation of the density of B. grandis and the use of fire to facilitate the breakdown of dead Banksia trunks in the Eucalyptus marginata (jarrah) forest may reduce the spread and impact of P. cinnamomi.     

Susceptibility of the Quercus rubra root system to Phytophthora cinnamomi; comparison with chestnut and other oak species

The susceptibility of oak seedlings (Quercus palustris, Quercus robur, Quercus rubra) and chestnut seedlings (Castanea sativa) to Phytophthora cinnamomi was tested. The dynamics of infection was examined in plant material raised in a rhizotron. In the oak species, primary root tissues were susceptible whereas secondary cortical tissues showed some resistance to P. cinnamomi. Secondary cortical tissues of the tap root in C. sativa were susceptible. Inoculations with P. cinnamomi were performed both in situ and on excised roots of mature Q. rubra. In both cases, the resistance of Q. rubra roots and shoots was negatively correlated with diameter at the inoculation point. Small roots (l–5-cm diameter) were resistant, whereas collar and trunk were susceptible. In contrast to oak, small excised roots of mature C. sativa (0.7–2-cm diameter) were susceptible to P. cinnamomi. This may explain why P. cinnamomi does not induce a decline of the attacked oaks, but rather a trunk canker.     

Relative susceptibility of oaks to seven species of Phytophthora isolated from oak forest soils

Isolates of Phytophthora cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercetorum and two unidentified species were tested for their pathogenicity to eastern US oak species by root and stem inoculations. Experiments were conducted during two different periods and included 1‐, 2‐ and 20‐year‐old oaks grown under greenhouse and field conditions. Species of Phytophthora were pathogenic in varying degrees to the oak species tested. All species were pathogenic to fine and taproots of at least one oak species. The fine root damage caused by the species of Phytophthora ranged from 9 to 55% when compared to the controls. Roots were more susceptible during the fall inoculation period than the summer. With exception of Phytophthora sp1 and P. quercina‐like, all species of Phytophthora were pathogenic to oak stems with P. cinnamomi and P. citricola being the most aggressive. Quercus montana and Q. rubra were the most susceptible oak species to stem inoculation. Lesion sizes were considerably larger when 20‐year‐old trees were inoculated. Generally, no significant differences in lesion sizes were detected in greenhouse tests when the summer and fall inoculation periods were compared. However, on 2‐year‐old field‐grown seedlings, lesion sizes were considerably smaller or not significantly different from controls during the fall inoculation period, suggesting lower, late season temperatures may restrict lesion development.     

Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees

Soil‐borne species of Phytophthora were isolated from 19 of 30 examined oak forest areas in Italy. The frequency of isolated Phytophthora spp. (35.2%) was significantly correlated with soil pH and longitude of the sites. Eleven Phytophthora species were detected. Phytophthora cambivora, P. cinnamomi and P. cactorum were recovered from sites in central and southern Italy whereas P. quercina was isolated in the northern and central part of the country. Phytophthora citricola occurred all over Italy. Phytophthora quercina was the only species significantly associated with declining oak trees.     

Phytophthora disease of Quercus ilex in south‐western Spain

Oak decline that was affecting three holm oak sites in the province of Huelva (south‐western Spain) was studied during 1998–1999. The syndromes of dieback and sudden death have been observed and, in both cases, foliar symptoms were associated with root rot. Characterization of the fungal isolates from necrotic roots led us to identify Phytophthora cinnamomi A2 as consistently associated with the disease. The optimum growth temperatures of these isolates were very high (30°C). Inoculation tests under controlled conditions demonstrated the pathogenicity of the isolates on holm and cork oak seedlings. None of the other biotic factors of Mediterranean oak decline that have been previously described were found in the present study and so, in this case, the forest decline model does not seem to be necessary in order to explain the disease observed. The defoliation and mortality of the oaks was primarily caused by P. cinnamomi, although some abiotic factors such as alternating periods of drought and wet weather in the region may play an important role.     

Calcium mineral nutrition increases the tolerance of Quercus ilex to Phytophthora root disease affecting oak rangeland ecosystems in Spain

Root rot caused by the soil-borne pathogen Phytophthora cinnamomi is leading to significant oak tree mortality in rangeland ecosystems in south western Spain. Susceptibility to P. cinnamomi infections of Q. ilex seedlings with a standard nutrition, deficient in K⁺, and deficient in Ca²⁺, was tested. Oaks deficient in K⁺ showed high values in Ca²⁺ content and were tolerant to the disease. Nutritional deficiency in Ca²⁺, however, did not lead to a higher K⁺ level in plants and induced poorer root development. In addition, K⁺ plant content does not appear to have any effect on pathogen tolerance. Based on these results, we conclude that satisfactory calcium nutrition may confer Holm oaks with a greater tolerance to root disease caused by P. cinnamomi. For this reason, limestone supplements are recommended as a measure against root rot caused by P. cinnamomi in rangelands in southern Spain, as a good option for control of oak root disease.     

Influence of site factors on the impact of Phytophthora cinnamomi in cork oak stands in Portugal

Although decline of cork (Quercus suber) and holm oak trees (Quercus rotundifolia) has been described in Portugal in the late years of the 19th century, its development has become a motive of high concern during the last two decades. The presence of Phytophthora cinnamomi in cork and holm oak stands was surveyed in four different regions of the country (Trás‐os‐Montes, Alentejo, Ribatejo and Algarve) during 1995–98. Tree decline severity, sudden death and site characteristics were assessed in 56 sites representing varied conditions. The pathogen was isolated from oak roots and rhizosphere samples in 27 of those places. Other plant species from natural vegetation were sampled in three active disease centres. This survey showed that 56% of the surveyed species of shrub flora were infected with P. cinnamomi, which was detected mainly on species belonging to the families Ericaceae, Cistaceae and Leguminosae. Recovery of P. cinnamomi was more frequent in shallow soils (Leptosols and complex Leptosols and Luvisols). These soils are more common in the south (Algarve), where decline has a high impact. Soils with low fertility and low mineral nutrient levels, particularly phosphorus, seemed to favour infection. Site aspect and topographic tree situation were also evaluated. Sites facing south showed higher occurrence of P. cinnamomi, which was also more frequent in slopes and valleys than on hilltops. In Algarve, a relationship could be established between the crown status and the presence of P. cinnamomi in roots and rhizosphere. Different morphotypes of P. cinnamomi could be distinguished in vitro, and their occurrence in the field was correlated with particular site characteristics. Further research needs and management strategies to limit the extension of the disease are discussed.     

Occurrence,incidence and associations among fungal pathogens and Agrilus auroguttatus,and their roles in Quercus agrifolia decline in California

Synchronous decline of oak (Quercus spp.) trees in woodlands has been described in Europe and eastern North America as a complex interaction of stressors that predispose, incite or contribute to tree death. This study presents a 2‐year (2010–2011) assessment of the role of pathogens in coast live oak (Quercus agrifolia) woodlands in southern California where oak mortality occurs in locations that are infested and uninfested by the goldspotted oak borer (GSOB, Agrilus auroguttatus). Cumulative coast live oak mortality was not significantly different between sites and was weakly correlated with Diplodia corticola and GSOB incidence and negatively correlated with annual relative humidity. Multiple logistic regression models explained the presence of individual fungi or GSOB at the tree level. Fisher's exact test analysis determined that the presence of D. corticola, Fusarium solani, Dothiorella iberica, Cryptosporiopsis querciphila and Diatrypella verrucaeformis were each related to origin of sample location on tree, and C. querciphila was additionally related to symptom type on the bole. Multiple linear regression models showed high correlation between environmental variables and plot‐level incidence of both GSOB and D. corticola. Disease incidence (DI) for D. corticola was highest in GSOB‐uninfested locations. Jaccard index of association (J) showed that D. corticola was negatively associated with the presence of GSOB, F. solani and C. querciphila. Results suggest that oak decline in California is an example of a complex syndrome involving strong regional differences in factors that are associated with the problem.     

Pathogenicity of three Phytophthora spp. causing late seedling rot of Quercus ilex ssp. ballota

Within a research project on quality of plants for forestation of agricultural lands, we studied the aetiology of a late seedling rot affecting holm oak (Quercus ilex ssp. ballota) in two forest nurseries in southern Spain. Major disease symptoms were foliage wilting and necrosis of feeder roots. Phytophthora cinnamomi, Phytophthora cryptogea and Phytophthora drechsleri were isolated from necrotic roots of holm oaks. Selected isolates of the three Phytophthora species were pathogenic to Quercus ilex ssp. ballota and Quercus suber seedlings in artificial inoculations. Soil flooding conditions were essential for infection and root rot development. There was no host specificity among the species, the isolates of P. cinnamomi being the most virulent in all inoculated plants. In these inoculations, Q. ilex ssp. ballota plants were more susceptible than those of Q. suber. This work is the first report of P. cinnamomi, P. drechsleri and P. cryptogea affecting Q. ilex ssp. ballota in forest nurseries.