Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

The evergreen holm oaks (Quercus ilex subsp. ilex and Q. ilex subsp. ballota) are the most representative tree species in the Iberian peninsula and the main tree species in oak‐rangeland ecosystems (dehesas). Oak decline in western, central and southern parts of Spain has been associated with root rot caused by Phytophthora cinnamomi for decades. However, Phytophthora species such as P.  quercina and P. psychrophila have recently been found associated with Quercus decline in eastern Spain where calcareous soils are predominant. Soil and root samples from two Quercus forests presenting decline symptoms in two different geographical areas in eastern Spain (Carrascar de la Font Roja and Vallivana) were analysed by amplicon pyrosequencing. Metabarcoding analysis showed Phytophthora species diversity, and revealed that an uncultured Phytophthora taxon, named provisionally Phytophthora taxon ballota, was the predominant species in both areas. In addition, a real‐time PCR assay, based on the pyrosequencing results, was developed for the detection of this uncultured Phytophthora taxon, and also for the detection of P. quercina. TaqMan assays were tested on soil and root samples, and on Phytophthora pure cultures. The new assays showed high specificity and were consistent with metabarcoding results. A new real‐time PCR protocol is proposed to evaluate the implication of different Phytophthora spp. in oak decline in eastern Spain.     

Histological methods to detect the clubroot pathogen Plasmodiophora brassicae during its complex life cycle

The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that lives in close relationship with its host cell. The roots of the host plants are colonized and the plant growth is altered upon infection. While shoots can be stunted and show wilt symptoms after longer infection periods, the root system is converted to a tumorous root tissue, called ‘clubroot’, by alterations of the plant growth promoting hormones auxin, cytokinin and brassinosteroid. Because the life cycle occurs largely within the host cells, this leads to dramatic changes in host root morphology and anatomy. Thus, the identification of the respective protist structures in the host tissue by microscopy is challenging. Different staining methods as well as fluorescence and electron microscopy of thin sections can reveal specific life stages of P. brassicae and can yield additional information on the changes in the host tissues concerning, for example, cell wall properties. In addition, promoter–reporter fusions, immunostaining methods and in situ hybridization techniques can be used to gain additional information on the changes in the host roots.     

Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance

Castanea sativa is susceptible to Phytophthora spp., a serious root pathogen causing ink disease, while C. crenata and C. mollissima show resistance to infection. Interspecific controlled crosses were established for introgression of resistance genes from the resistant species into the susceptible C. sativa, and two mapping populations were created. Phytophthora cinnamomi resistance of each progeny was evaluated by root and excised shoot inoculation tests. The number of days of survival after root inoculation was the best discriminator of resistance to P. cinnamomi while the percentage of shoots with internal lesions was the symptom most associated with survival. The lesion progression rate in the excised shoot inoculation test was strongly and negatively correlated with survival in the root inoculation test. The excised shoot inoculation test appears to be a reliable approach for screening the resistance of chestnut genotypes to P. cinnamomi. Strong genetic correlations were obtained between survival and ink disease symptoms and among symptoms, indicating that common or linked genes might influence resistance to P. cinnamomi. The most resistant genotypes selected from this study will be tested for other commercial variables, such as ease of vegetative propagation and stock–scion compatibility.     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

Pathogenicity of 21 newly described Phytophthora species against seven Western Australian native plant species

The pathogenicity of some Phytophthora species recently described from Western Australia, together with P. cinnamomi as a control, was tested against seven Western Australian native plant species in the glasshouse. Host species were Banksia grandis, B. littoralis, B. occidentalis, Casuarina obesa, Corymbia calophylla, Eucalyptus marginata and Lambertia inermis. Twenty‐two Phytophthora species were grown on a vermiculite, millet seed and V8 substrate and used as soil inoculum when the plant hosts were approximately 3 months old. Pathogenicity was assessed after 6 weeks and plants were scored for death, root damage, and percentage reduction of shoot growth compared with control plants. The pathogenicity of P. cinnamomi was confirmed. Phytophthora niederhauserii was shown to be similar to P. cinnamomi in pathogenicity and of concern ecologically. Other species that killed one or more hosts were P. boodjera, P. constricta, P. elongata, P. moyootj and P. rosacearum, while P. condilina, P. gibbosa, P. gregata, P. litoralis and P. ‘personii’ caused significant reduction to shoot and/or root growth, but did not kill plants. Host species susceptible to the highest number of Phytophthora species were B. grandis, B. littoralis, B. occidentalis and E. marginata. No Phytophthora species tested killed C. calophylla.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

Comparison of root and foliar applications of potassium silicate in potentiating post‐infection defences of melon against powdery mildew

The application of silicon to the roots or leaves reduces the severity of powdery mildew (Podosphaera xanthii) in melon but the latter treatment is less effective. This study compared key biochemical defence responses of melon triggered by P. xanthii after root or foliar treatment with potassium silicate (PS). Treatments consisted of pathogen‐inoculated or mock‐inoculated plants supplied with PS via roots or foliarly, as well as a non‐treated control. The activity of defence enzymes and the concentration of phenolic compounds, lignin and malondialdehyde were determined from leaf samples at different time points after inoculation. Pathogen‐inoculated plants irrigated with PS showed both an accumulation of silicon and primed defence responses in leaves that were not observed in pathogen‐inoculated plants either sprayed with PS or not treated. These responses included the anticipated activity of peroxidase and accumulation of soluble phenols, the activation of chitinase and repression of catalase, and the stronger activation of superoxide dismutase, peroxidase and β‐1,3‐glucanase. Moreover, the lignin concentration increased in response to inoculation, whereas the malondialdehyde concentration decreased. For the foliar treatment, however, only an increase in lignin deposition was observed compared with the control plants. The results show that silicon strongly plays an active role in modulating the defence responses of melon against P. xanthii when supplied to the roots as opposed to the foliage.     

Age‐related susceptibility of Eucalyptus species to Phytophthora boodjera

Phytophthora boodjera is a newly described pathogen causing damping off and mortality of Eucalyptus seedlings in Western Australian nurseries. This study evaluated the age‐related susceptibility of several taxa of mallee Eucalyptus to P. boodjera in sterilized washed river sand‐infestation pot trials. Phytophthora cinnamomi and P. arenaria were included for comparison. Seedlings of Eucalyptus taxa were inoculated at 0, 2, 4, 12 and 88 weeks with individual Phytophthora isolates. Pre‐emergent mortality in the presence of Phytophthora was almost 100%. Post‐emergent mortality was 50–100%, depending on isolate, compared to 0% for the control. Mortality was also high for inoculated 1 month‐old seedlings (46–68%) and root length of surviving seedlings was severely reduced. Death from root infection was not observed for seedlings inoculated at 12 and 88 weeks, but they developed root necrosis and reduced root dry weight compared to non‐inoculated controls. Phytophthora boodjera is a pre‐ and post‐emergent pathogen of mallee eucalypts. These eucalypts are susceptible to P. boodjera at all life stages tested, but the mortality rates declined with plant age. Similar results were obtained for P. cinnamomi and P. arenaria. The events leading to its recent appearance in the nurseries remain unknown and further investigations are underway to determine if this is an introduced or endemic pathogen. The approach used here to understand the impact of a Phytophthora species on multiple hosts at different seedling ages is novel and sets a benchmark for future work.     

Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.     

Annual and herbaceous perennial native Australian plant species are symptomless hosts of Phytophthora cinnamomi in the Eucalyptus marginata (jarrah) forest of Western Australia

Resistant annual and herbaceous perennial plant species were identified as key hosts which allow Phytophthora cinnamomi to persist on severely impacted black gravel sites within the Eucalyptus marginata (jarrah) forest of southwest Western Australia. Of the annual and herbaceous perennial plant species present on black gravel sites, 15 out of 19 species were found to be hosts of P. cinnamomi, and 10 of these were symptomless hosts. In particular, the native annual Trachymene pilosa and the two native herbaceous perennials Stylidium diuroides and Chamaescilla corymbosa were commonly found to be hosts of the pathogen. Species from 12 new genera including three from new families (Crassulaceae, Droseraceae and Primulaceae) are reported for the first time to be hosts of P. cinnamomi. The species from which P. cinnamomi was recovered were the native species: Chamaescilla corymbosa, Crassula closiana, Drosera erythrorhiza, Hydrocotyle callicarpa, Levenhookia pusilla, Paracaleana nigrita, Podotheca angustifolia, Pterochaeta paniculata, Rytidosperma caespitosum, Siloxerus multiflorus, Stylidium diuroides and Trachymene pilosa, and the introduced annual weeds Hypochaeris glabra, Lysimachia arvensis and Pentameris airoides.     

Differences in constitutive and inducible defences in pine species determining susceptibility to pinewood nematode

The pinewood nematode, Bursaphelenchus xylophilus, originating from North America (NA), is a major invasive pine pest in Eurasia. It was first detected in Portugal in 1999 associated with maritime pine, Pinus pinaster, and has been differently affecting the main local pine species, P. pinaster and P. pinea. Field studies and direct inoculation experiments in Pinus spp. seedlings, under controlled conditions, were performed to assess whether the differences in constitutive and inducible defences are determining the different susceptibility of pine host species to B. xylophilus. Host co‐evolution with the pathogen was also assessed, including the NA P. radiata, widely used in forestry in the northeast of the Iberian peninsula. Pine mortality in the field was positively related with the abundance of B. xylophilus, and concentration of phenolics and condensed tannins in pines. In the greenhouse assay, seedling tissues were analysed for constitutive investment in defences, as well as the potential inducibility of those defences as driven by B. xylophilus inoculation. Slower growing P. pinea presented higher levels of constitutive defences than faster growing P. pinaster, with only P. pinaster being affected by B. xylophilus. Furthermore, co‐evolution with the pathogen is important, with the fast‐growing NA P. radiata presenting an inducible and effective response to B. xylophilus. Results point to the importance of integrating data on pine life history traits, including growth rate, and production of constitutive and inducible defences, into predictive models for this invasive forest pest.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included wild olive clones Ac‐4 and Ac‐15, olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138I‐YFP, the latter being obtained in this study. In planta observations indicated that V138I‐YFP colonizes the roots and stems of the olive and wild olive genotypes, and that GFP22 grows endophytically within the roots of them all. YFP fluorescence signal quantifications showed that: (i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being higher in Ac‐15 than in Ac‐4 plants; and (ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.     

Production of avocado trees infected with Phytophthora cinnamomi under different management regimes

Avocado root rot is the most important disease of this fruit crop worldwide. This pathology may be caused by several biotic and abiotic agents, with the oomycete Phytophthora cinnamomi being the pathogen more frequently associated with poor phytosanitary conditions. There are disease control methods available that can reduce disease severity and allow plants to recover; however, they are not consistently and promptly applied. In addition, only chemical products are used by farmers as the preferred management method. This research aimed to evaluate different root rot management strategies in a commercial orchard. Data suggest that individual control methods are not as effective as when they are applied in combination, as in the T8 treatment (metalaxyl + mancozeb applied in drench; injection of potassium phosphite to each plant stem; potassium silicate applied in drench; addition of a layer of organic mulch and incorporation of 10 kg of composted substrate, both applied to the ground around the base of each tree). Using this strategy, the area under the disease progress curve for the avocado root rot was reduced by up to 68.6%, and the extra‐quality avocado fruit class increased by as much as 44% compared to the diseased control plants (T0) (P < 0.01). With the combined treatment T8, farm income showed a 9.5‐fold increase, probably due to an increase in the percentage of viable roots by up to 9.4‐fold, which would have improved nutrient and water uptake.     

Differential behaviour of sheath blight pathogen Rhizoctonia solani in tolerant and susceptible rice varieties before and during infection

This study characterized the early infection and establishment of the sheath blight pathogen Rhizoctonia solani on a tolerant rice variety, Swarnadhaan (IET 5656), and a susceptible variety, Swarna (MTU 7029). Assays using whole plants showed that disease severity was higher in Swarna than Swarnadhaan. In a detached leaf assay, Swarnadhaan showed a disease index that was 50% less than that with Swarna. Rhizoctonia solani exhibited different growth behaviour in the tolerant and susceptible varieties. The pathogen showed more hyphal growth in the susceptible host than in the tolerant variety. It also showed profuse branching, making intimate contact with the host surface to form more inter‐ and intracellular structures, and greater sclerotial development in the susceptible host compared to the tolerant one. Using light and scanning electron microscopy, it was observed for the first time that the pathogen could intercept host surface structures and use these for anchorage or penetration. Transformed R. solani, expressing green fluorescent protein, was observed using confocal laser scanning microscopy to investigate pathogen behaviour, including the formation of infection cushions and subsequent colonization of the host tissues. This is the first ultrastructural report to characterize the differential behaviour of the sheath blight pathogen in the vicinity and within tolerant and susceptible rice plants.     

Pineapple heart rot isolates from Ecuador reveal a new genotype of Phytophthora nicotianae

Pineapple heart rot disease, caused by Phytophthora nicotianae (syn. P. parasitica), is responsible for significant annual reductions in crop yield due to plant mortality. In Ecuador, new infections arise during the rainy season and increase production costs due to the need for biocontrol and fungicide applications. Studies of P. nicotianae population structure suggest that certain genetic groups are associated with host genera; however, it is not clear how many host‐specific lineages of the pathogen exist or how they are related. The objectives of this study were to determine the level of genetic variation in the P. nicotianae population causing heart rot disease of pineapple in Ecuador and compare the genotypes found on pineapple to those previously reported from citrus, tobacco and ornamentals. Thirty P. nicotianae isolates collected from infected pineapple leaves from four farms were genotyped using nine simple sequence repeat loci. In addition, the DNA sequences of mitochondrial loci cox2 + spacer and trnG‐rns were analysed. Together, these loci supported a single clonal lineage with two multilocus genotypes differing in a single allele and low mitochondrial diversity. This lineage was distinct but closely related to isolates collected from vegetables and ornamentals in Italy. The results support the hypothesis of host specialization of P. nicotianae in intensive cropping systems and contribute to the understanding of population structure of this important pathogen.     

Penetration of suberized periderm of a woody host by Phytophthora cinnamomi

The mechanisms by which Phytophthora cinnamomi zoospores infect inundated, above‐ground woody stem tissue are described. Using 4–6‐ and 18‐month‐old jarrah seedlings, the infection courts were identified and the invasion of the stems at sites of zoospore cyst binding were described. Stems were inoculated with a suspension of motile zoospores on the green stem/young periderm region. Light microscopy was used to examine penetration at sites of taxis, and fluorescent microscopy was used to examine penetration sites of seedlings with intact periderm. Two main infection courts were identified on stems: the emerging axillary shoot and the region of stem immediately surrounding an axillary shoot, where the periderm was thin or discontinuous. Invasion also occurred at sites where the developing shoot had not yet emerged but was at the stem surface. At these sites the pathogen also directly invaded through the thin‐walled phellem of the periderm surrounding the shoot. Zoospores of P. cinnamomi were not attracted to stomata on mature leaves or green stems. Penetration of the epidermal cell layer of the axillary bud leaf primordia was inter‐ and intra‐cellular; growth of hyphae in the periderm surrounding the shoot was intercellular; while in collenchyma it was inter‐ and intra‐cellular, being intercellular between polyphenolic‐rich cells. Exposed stem collenchyma was also directly invaded immediately adjacent to the young axillary shoot. Zoospores demonstrated taxis to sites of discontinuous periderm, similar to wounded areas where the outer protective layers of the plant are breached. This study presents the first evidence that P. cinnamomi is capable of intercellular penetration of suberized periderm.     

Investigations into Encephalartos insect pests and diseases in South Africa and identification of Phytophthora cinnamomi as a pathogen of the Modjadji cycad

South Africa holds the greatest diversity of Encephalartos species globally. In recent years several reports have been received of Encephalartos species in the country dying of unknown causes. The aim of this study was to investigate the presence of, and identify the causal agents of, diseases of Encephalartos species in the Gauteng and Limpopo Provinces of South Africa. Plant material with symptoms and insects were collected from diseased plants in private gardens, commercial nurseries and conservation areas in these regions. Insects collected were identified based on morphology, and microbial isolates based on morphology and DNA sequence data. Insect species identified infesting cultivated cycads included the beetle Amorphocerus talpa, and the scale insects Aonidiella aurantii, Aspidiotus capensis, Chrysomphalus aonidum, Lindingaspis rossi, Pseudaulacaspis cockerelli, Pseudaulacaspis pentagona and Pseudococcus longispinus. Fungal species isolated from diseased plants included species of Diaporthe, Epicoccum, Fusarium, Lasiodiplodia, Neofusicoccum, Peyronellaea, Phoma, Pseudocercospora and Toxicocladosporium. The plant pathogen Phytophthora cinnamomi was identified from E. transvenosus plants in the Modjadji Nature Reserve. Artificial inoculation studies fulfilled Koch's postulates, strongly suggesting that P. cinnamomi is responsible for the deaths of these plants under field conditions.     

Implication of peroxynitrite in defence responses of potato to Phytophthora infestans

In this study peroxynitrite (ONOO^?) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO^? formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post‐inoculation (hpi). The early ONOO^? over‐accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO^? formation; however, an enhanced level of NO and superoxide correlated with TPx up‐regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over‐accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin‐like protease‐like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO^? followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up‐regulation of the key defence markers, including the PR‐1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin‐dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO^?, triggering defence responses.