Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

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.     

Association of ‘Candidatus Liberibacter asiaticus’ root infection,but not phloem plugging with root loss on huanglongbing‐affected trees prior to appearance of foliar symptoms

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem‐limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem‐limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem‐plugging as previously hypothesized. The role of root infection in systemic insect‐vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem‐limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem‐limited bacteria to gain greater understanding of how these pathogens cause disease and spread.     

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.     

Ralstonia solanacearum virulence in tomato seedlings inoculated by leaf clipping

Ralstonia solanacearum is a phytopathogenic bacterium that colonizes the xylem vessels of host plants leading to a lethal wilt disease. Although several studies have investigated the virulence of R. solanacearum on adult host plants, infection studies of this pathogen on the seedling stages of hosts are less common. In a preliminary observation, inoculation of R. solanacearum F1C1 on 6‐ to 7‐day‐old tomato seedlings by a simple leaf‐clip strategy resulted in a lethal pathogenic condition in seedlings that eventually killed these seedlings within a week post‐inoculation. This prompted testing of the effect of this inoculation technique in seedlings from different cultivars of tomato and similar results were obtained. Colonization and spread of the bacteria throughout the infected seedlings was demonstrated using gus‐tagged R. solanacearum F1C1. The same method of inoculating tomato seedlings was used with R. solanacearum GMI1000 and independent mutants of R. solanacearum GMI1000, deficient in the virulence genes hrpB, hrpG, phcA and gspD. Wildtype R. solanacearum GMI1000 was found to be virulent on tomato seedlings, whereas the mutants were found to be non‐virulent. This leaf‐clip technique, for inoculation of tomato seedlings, has the potential to be a valuable approach, saving time, space, labour and costs.     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Severity of phytophthora root rot and pre‐emergence damping‐off in subterranean clover influenced by moisture,temperature, nutrition,soil type,cultivar and their interactions

Studies were carried out in controlled environment rooms reflecting field situations. In the presence of the devastating soilborne pathogen Phytophthora clandestina, subterranean clover (Trifolium subterraneum) seedling emergence was significantly affected by moisture, soil type, temperature and cultivar. The level of rotting of tap and lateral roots was significantly affected by nutrition, soil type, temperature and cultivar. There were significant interactions involving temperature, moisture, soil type and cultivar; cultivar resistance, high moisture, high or medium temperature, high nutrition and sand soil all contributed towards less pre‐emergence damping‐off and tap and lateral root disease and to greater clover productivity. Host resistance of subterranean clover cultivars was critical for reducing disease severity and increasing productivity, even when favourable environmental conditions for severe disease occurred. In the presence of P. clandestina, the most resistant cultivar, Seaton Park, performed best under a high temperature, high nutrition and high moisture combination, but showed lower productivity under conditions of low nutrition or lower temperature, even when moisture level was high. In contrast, less resistant cultivars Riverina and Meteora had less disease and greater productivity under low moisture conditions. Findings reflect field observations that pre‐emergence damping‐off and root disease from P. clandestina in subterranean clover is particularly severe under colder conditions and in nutritionally impoverished sandy soils, and demonstrate how variations in soil type, nutrition, moisture, temperature and cultivar have profound effects on the expression and severity of phytophthora pre‐emergence damping‐off and root disease and the productivity of subterranean clover forages.     

Arabidopsis thaliana,an experimental host for tomato yellow leaf curl disease‐associated begomoviruses by agroinoculation and whitefly transmission

Tomato yellow leaf curl disease is one of the most devastating viral diseases affecting tomato crops worldwide. This disease is caused by several begomoviruses (genus Begomovirus, family Geminiviridae), such as Tomato yellow leaf curl virus (TYLCV), that are transmitted in nature by the whitefly vector Bemisia tabaci. An efficient control of this vector‐transmitted disease requires a thorough knowledge of the plant–virus–vector triple interaction. The possibility of using Arabidopsis thaliana as an experimental host would provide the opportunity to use a wide variety of genetic resources and tools to understand interactions that are not feasible in agronomically important hosts. In this study, it is demonstrated that isolates of two strains (Israel, IL and Mild, Mld) of TYLCV can replicate and systemically infect A. thaliana ecotype Columbia plants either by Agrobacterium tumefaciens‐mediated inoculation or through the natural vector Bemisia tabaci. The virus can also be acquired from A. thaliana‐infected plants by B. tabaci and transmitted to either A. thaliana or tomato plants. Therefore, A. thaliana is a suitable host for TYLCV–insect vector–plant host interaction studies. Interestingly, an isolate of the Spain (ES) strain of a related begomovirus, Tomato yellow leaf curl Sardinia virus (TYLCSV‐ES), is unable to infect this ecotype of A. thaliana efficiently. Using infectious chimeric viral clones between TYLCV‐Mld and TYLCSV‐ES, candidate viral factors involved in an efficient infection of A. thaliana were identified.     

Root infection of sugar beet by <Emphasis Type="Italic">Cercospora beticola</Emphasis> in a climate chamber and in the field

Sugar beet root infection by Cercospora beticola, the causal agent of Cercospora leaf spot (CLS), was studied in a climate chamber and in the field. In the climate chamber, root incubation of susceptible seedlings with a conidial suspension resulted in disease incidences that were significantly different for two sugar beet cultivars (Auris: 0.8 ± 0.14 and A00170: 0.5 ± 0.18; P < 0.05) with regard to the control treatment 35 days after root incubation in a standard potting soil-fine river sand mixture. In a field trial with susceptible cv. Savannah with soil-incorporated CLS-infested leaf material, disease developed four weeks earlier in the infested plots than in the control plots. The probability that disease develops in the field was significantly higher for the infested than for the control plots (P < 0.05). Symptomless plants from infested field plots transferred to the glasshouse to induce leaf spot symptoms showed a significantly higher probability to induce symptom development (0.4 ± 0.08), than plants from control plots (0.02 ± 0.02) (P <0.05) 14 days after transfer. This probability was significantly higher than for plants that remained in three of the infested field plots (0.2 ± 0.04; 0.2 ± 0.05 and 0.2 ± 0.04 respectively), except for one infested field plot (0.4 ± 0.05) on July 5. We conclude that C. beticola is able to infect sugar beet seedlings through their roots and that latent CLS infections in sugar beet lead to symptom development at high temperatures (> 20 °C) and high relative humidity (> 95) in our climate chamber or after canopy closure in the field. Quantification of root infection and long term survival in soil is necessary to assess its contribution to the epidemiology and life cycle of Cercospora beticola. Cultural methods such as a wider crop rotation, management of crop debris and ploughing systems may provide control strategies alternative to or reducing fungicide input.     

Non‐pathogenic rhizosphere bacteria belonging to the genera Rhizorhapis and Sphingobium provide specific control of lettuce corky root disease caused by species of the same bacterial genera

Lettuce corky root (CR) is caused by bacteria in the genera Rhizorhapis, Sphingobium, Sphingopyxis and Rhizorhabdus of the family Sphingomonadaceae. Members of this family are common rhizosphere bacteria, some pathogenic to lettuce. Sixty‐eight non‐pathogenic isolates of bacteria obtained from lettuce roots were tested for control of CR caused by Rhizorhapis suberifaciens CA1^T and FL1, and Sphingobium mellinum WI4^T. In two initial screenings, 10 isolates significantly reduced CR induced by one or more pathogenic strains on lettuce seedlings in vermiculite, while seven non‐pathogenic isolates provided significant CR control in natural or sterilized field soil. Rhizorhapis suberifaciens FL11 was effective at controlling all pathogenic strains, but most effective against R. suberifaciens CA1^T. The other selected isolates controlled only pathogenic strains belonging to their own genus. In a greenhouse experiment, a soil drench with selected biocontrol agents (R. suberifaciens FL11, Sphingomonas sp. NY3 and S. mellinum CA16) controlled CR better than seed treatments or application of alginate pellets. In microplots infested with R. suberifaciens CA1^T, seed treatment with R. suberifaciens FL11 provided complete control and a soil drench with FL11 significantly reduced the disease. Pathogenicity tests with FL11 on 23 plant species in 10 families resulted in slight yellowing on roots of lettuce and close relatives; similar yellowing appeared on some roots of non‐inoculated lettuce plants. This research showed that biocontrol agents can be genus‐specific. Only one isolate, FL11, provided more general control of various pathogenic strains causing CR even in field soil in pots and microplots.     

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.     

Nematicidal activity of Ochradenus baccatus against the root‐knot nematode Meloidogyne javanica

Ochradenus baccatus is a widely distributed shrub in desert regions of the Middle East and North Africa. This plant's nematicidal activity against the root‐knot nematode Meloidogyne javanica was evaluated because it has been found to contain exceptionally high levels of glucosinolates. In in vitro assays with aqueous extracts of the plant, 100% of second‐stage juveniles were immobilized after exposure to 4% root‐core extract for 48 h; 8% root‐core extract suppressed their hatching by 87%, whereas stem, flower and root bark showed lower activity. Incorporation of root core or bark into the soil, as fresh or dry powder at 1 and 0·5% (w/w), respectively, reduced the number of nematodes recovered from the soil by 95–100%, whereas the flower and stem were much less effective. Results from further pot experiments indicated that only the root bark consistently contains nematicidal compounds which are effective in soil, whereas the nematicidal activity of the root core in soil was inconsistent. The presence of non‐volatile lipophilic and lipophobic nematicidal compounds in the root bark was suggested by extraction with different polar solvents, but these compounds do not seem to be isothiocyanates – glucosinolate‐hydrolysed compounds with nematicidal activity. Very poor host status of Ochradenus baccatus to M. javanica, M. incognita and M. hapla, but with root‐penetration rates of juveniles similar to those in tomato roots, suggest that this plant may be used as a cover plant or trap plant to reduce nematode populations in the soil.     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

Survival of Phytophthora ramorum in Rhododendron root balls and in rootless substrates

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium. Following inoculation of the root balls, the aboveground plant parts stayed symptomless, whilst the pathogen could be recovered with a novel non‐destructive baiting assay from the root balls until at least 8 months post‐inoculation. Plating of surface‐sterilized roots and direct microscopic analysis confirmed the presence of P. ramorum in the roots. Phytophthora ramorum could also be baited from the root balls of symptomless Rhododendron plants from commercial nurseries, even 2 years after acquisition. Survival of P. ramorum in rootless media was assessed after burying disks of infected leaf material below the soil surface in columns filled with four different undisturbed forest substrates or a potting medium, and incubated at an outdoor quarantine facility. Phytophthora ramorum could be recovered at least 33 months after burial from all substrates, with a significant increase in recovery after the winter period. These data suggest the possibility for long‐term symptomless presence of P. ramorum in root balls of commercial Rhododendron plants as well as survival in potting medium and different forest substrates under western European climate conditions. Symptomless presence in root balls can contribute to latent spread of this pathogen between nurseries. The novel baiting test, being non‐destructive, simple and applicable to a relatively large number of plants, can offer a valuable tool to test plants for the presence of Phytophthora species in root balls.     

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.     

Seasonal susceptibility of citrus scions to <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> and <Emphasis Type="Italic">P. nicotianae</Emphasis> and the influence of environmental and host-linked factors on infection development

Three citrus scions were evaluated to determine seasonal changes in susceptibility to infections by Phytophthora citrophthora and Phytophthora nicotianae. In a period of 24 months, the Clementine mandarin cv. Hernandina, the hybrid Fortune mandarin and the sweet orange cv. Lane-Late were branch-inoculated under field and laboratory conditions. Field studies showed that the cultivars inoculated with P. citrophthora developed the highest lesion areas during March–June (spring) and September–October (autumn) and with P. nicotianae from June to August (summer). However, lesion areas on detached citrus branches did not show a definite pattern of infection because lesion sizes fluctuated irregularly during the study. The lesion area caused by P. nicotianae in different citrus scions correlated significantly with the monthly mean maximum values of temperature, relative humidity, and the percentage of the relative water content in the 24-month period of inoculations. In contrast, there was no correlation between these variables and the extent of colonisation by P. citrophthora. Nevertheless, a significant relationship was observed between lesion areas caused by P. citrophthora from October to May of each year and the same variables that were significant in inoculations with P. nicotianae. Seasonal changes in the susceptibility of citrus cultivars to P. citrophthora and P. nicotianae may facilitate timing of disease control measures to coincide with periods when disease development is greatest.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.