Contrasting canopy and fibrous root damage on Swingle citrumelo caused by ‘Candidatus Liberibacter asiaticus’ and Phytophthora nicotianae

Huanglongbing (HLB), associated with the phloem‐limited bacterium ‘Candidatus Liberibacter asiaticus’ (Las), is devastating trees in citrus orchards of Florida. Additionally, Phytophthora nicotianae, omnipresent in citrus soils, causes root rot that reduces water and nutrient uptake by fibrous roots. To investigate fibrous root damage and replacement and canopy size in relation to infection of fibrous roots by Las and P. nicotianae, rootstock seedlings of Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) were inoculated with Las or P. nicotianae in two greenhouse pot trials. Phytophthora nicotianae caused root damage within 5 weeks post‐inoculation, which led to greater reduction of canopy size than for Las‐infected seedlings by the end of the experiment. Las increased accumulation of fibrous root biomass at 5 weeks post‐root trimming (wpt) in the 2014 trial and at 11 wpt in the 2015 trial. New root length was not consistently increased by Las. Reduced total leaf area of symptomless Las‐infected seedlings compared to noninoculated controls might be due to the combined effect of altered carbohydrate allocation between shoots and roots and altered leaf morphology.     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

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.     

Effect on Resistance to Tylenchulus semipenetrans of Hybrid Citrus Rootstocks Subjected to Continuous Exposure to High Population Densities of the Nematode

Experiments simulating interplanting of resistant rootstocks with susceptible rootstocks that maintain high population densities of Tylenchulus semipenetrans in field soil were carried out in microplots at two locations, and in an naturally infested orchard. Selections of Cleopatra mandarin (03) × Poncirus trifoliata (01) 03.01.5 and 03.01.13, Citrus volkameriana (23) × P. trifoliata 23.01.17, Troyer citrange (02) × Cleopatra mandarin 02.03.24, Troyer citrange × Common mandarin (04) 02.04.18, King mandarin (05) × P. trifoliata 05.01.7, and Carrizo citrange were exposed to continuous high population densities of a population of the Mediterranean biotype of T. semipenetrans. The selection 23.01.17 retained its resistance in the microplots and in the field (< 1.2% females and eggs per gram fibrous root of those on Carrizo citrange). The selection 03.01.5 also retained its resistance in the microplots at Moncada (< 0.5% females and eggs per gram fibrous root of those on Carrizo citrange) but numbers of females and eggs per gram fibrous root were 27% and 22% at Amposta, and 139% and 18% in the orchard of those on Carrizo citrange, respectively. The selection 05.01.7 supported equal number of females and 43% eggs per gram fibrous root of those on Carrizo citrange in the nematode-infested orchard. The remaining selections supported high populations of T. semipenetrans.     

Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo

Citrus canker (caused by the bacterial pathogen Xanthomonas citri subsp. citri, Xcc) can cause severe damage to citrus. It is endemic in Florida, and occurs in other citrus growing regions. The bacterium is dispersed predominantly in rain splash. To simulate dispersal in splash, and to investigate the effect of wind speed on infection, young plants of Swingle citrumelo were exposed to sprayed inoculum at different wind speeds. Wind was generated using an axial fan, and a pressurized sprayer delivered the inoculum spray. In the five experiments, higher wind speeds (>10 m s⁻¹) consistently resulted in higher incidence and severity of citrus canker developing. By 15 ms⁻¹, there was a dramatic increase in disease. Visible injury to leaves of Swingle citrumelo due to wind was evident at wind speeds ≥ 13 m s⁻¹. The relationship between wind speed and disease, and wind speed and injury was described by a logistic model. More disease was associated with visible injury as the wind speed increased, and disease not associated with visible injury also increased with wind speed. The petiole-leaflet junction was more often infected at higher wind speeds (≥17 m s⁻¹). The concentration of the Xcc inoculum increased the incidence and severity of citrus canker in all experiments. Reducing wind speed in citrus groves with the aid of wind breaks may contribute to a reduction in the severity of an epidemic by reducing dispersal and infection events.     

Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum

The effect of soil solarization and Trichoderma harzianum on induced resistance to grey mould (Botrytis cinerea) and powdery mildew (Podosphaera xanthii) was studied. Plants were grown in soils pretreated by solarization, T. harzianum T39 amendment or both, and then their leaves were inoculated with the pathogens. There was a significant reduction in grey mould in cucumber, strawberry, bean and tomato, and of powdery mildew in cucumber, with a stronger reduction when treatments were combined. Bacillus, pseudomonad and actinobacterial communities in the strawberry rhizosphere were affected by the treatments, as revealed by denaturing gradient gel electrophoresis fingerprinting. In tomato, treatments affected the expression of salicylic acid (SA)‐, ethylene (ET)‐ and jasmonic acid (JA)‐responsive genes. With both soil treatments, genes related to SA and ET – PR1a, GluB, CHI9 and Erf1 – were downregulated whereas the JA marker PI2 was upregulated. Following soil treatments and B. cinerea infection, SA‐, ET‐, and JA‐related genes were globally upregulated, except for the LOX genes which were downregulated. Upregulation of the PR genes PR1a, GluB and CHI9 in plants grown in solarized soil revealed a priming effect of this treatment on these genes' expression. The present study demonstrates the capacity of solarization and T. harzianum to systemically induce resistance to foliar diseases in various plants. This may be due to either a direct effect on the plant or an indirect one, via stimulation of beneficial microorganisms in the rhizosphere.     

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.     

Concentration-dependent effect of salicylic acid application on wheat seedling resistance to take-all fungus, <Emphasis Type="Italic">Gaeumannomyces graminis</Emphasis> var. <Emphasis Type="Italic">tritici</Emphasis>

Application of 0.1 and 0.2 mM salicylic acid (SA) significantly reduced take-all disease caused by Gaeumannomyces graminis var. tritici (Ggt) and increased the root and shoot lengths and biomass, whereas 0.5 and 1 mM SA had no significant effect. The effective SA concentrations also increased the activities of soluble peroxidase (SPOX) and cell-wall-bound peroxidase (CWPOX) and the concentration of total phenolic compounds. SPOX activity was highest at days 4 and 3 in healthy roots and those inoculated with Ggt, respectively, and that of CWPOX at day 6 in both healthy and inoculated roots. The concentration of phenolic compound was also highest at day 3 in both healthy roots and those inoculated with Ggt. The results indicate that the protective effect of SA depends on certain concentrations which increase peroxidase activity and phenolic compounds accumulation in the wheat roots; higher SA concentrations did not differ from the controls.     

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.     

Suppressive effect of abscisic acid on systemic acquired resistance in tobacco plants

Recent studies have indicated that the phytohormone abscisic acid (ABA), induced in response to a variety of environmental stresses, plays an important role in modulating diverse plant–pathogen interactions. In Arabidopsis thaliana, we previously clarified that ABA suppressed the induction of systemic acquired resistance (SAR), a plant defense system induced by pathogen infection through salicylic acid (SA) accumulation. We investigated the generality of this suppressive effect by ABA on SAR using tobacco plants. For SAR induction, we used 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) that activate upstream and downstream of SA in the SAR signaling pathway, respectively. Wild-type tobacco plants treated with BIT or BTH exhibited enhanced disease resistance against Tobacco mosaic virus (TMV) and tobacco wildfire bacterium, Pseudomonas syringae pv. tabaci (Pst), however, which was suppressed by pretreatment of plants with ABA. Pretreatment with ABA also suppressed the expression of SAR-marker genes by BIT and BTH, indicating that ABA suppressed the induction of SAR. ABA suppressed BTH-induced disease resistance and pathogenesis-related (PR) gene expression in NahG-transgenic plants that are unable to accumulate SA. The accumulation of SA in wild-type plants after BIT treatment was also suppressed by pretreatment with ABA. These data suggest that ABA suppresses both upstream and downstream of SA in the SAR signaling pathway in tobacco.     

Jasmonic acid and abscisic acid play important roles in host–pathogen interaction between Fusarium graminearum and wheat during the early stages of fusarium head blight

The effect of phytohormones on the defense response of wheat against Fusarium graminearum infection was investigated. Infection of heads with F. graminearum induced accumulation of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and indole acetic acid (IAA). Exogenous phytohormone treatments showed crosstalk between them and a complex effect on expression of the genes ATB2, ExpB6, LEA Td16, PR1, Pdf1.2, PR4. JA treatment reduced F. graminearum growth and fusarium head blight (FHB) symptoms while an increase in FHB was observed with ABA. Transient down-regulation of allene oxide synthase (AOS) supports a complex role for JA in wheat head.     

Exogenous application of melatonin improves plant resistance to virus infection

In this study, melatonin (MEL)-mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti-virus infection activity to 37.4% in virus-infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT-PCR and virus titres measured by dot-ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT-PCR, in virus-infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H₂O₂) in the virus-infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL-induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT-PCR and virus titres analysed by dot-ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL-mediated plant resistance to TMV is proposed. MEL-mediated plant resistance to viruses provides a new avenue to control plant viral diseases.     

Effects of pymetrozine, an antifeedant of Hemiptera, on Asian citrus psyllid, Diaphorina citri, feeding behavior, survival and transmission of Candidatus Liberibacter asiaticus

BACKGROUND: Vector‐borne plant disease management can be enhanced by deployment of antifeedants in addition to the use of broad‐spectrum neurotoxic insecticides. The effects of pymetrozine on Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, feeding behaviour, survival and transmission of Candidatus Liberibacter asiaticus (Las), the presumed causal pathogen of huanglongbing, were investigated. RESULTS: Pymetrozine applied at 52 and 104 µg mL^?1 to citrus plants [Swingle citrumelo (X Citroncirus webberi Ingram and Moore)] modified the feeding behavior of ACP and increased the amount of time spent performing non‐penetration behaviors while decreasing the time spent performing ingestion behaviors compared with the controls 1 day after treatment. However, the antifeedant effect of pymetrozine subsided 5 days after application. Pymetrozine reduced the survival of both adults and nymphs on treated plants compared with the control. However, it had a greater impact on survival of nymphs than on survival of adults. Pymetrozine applied at 52 and 104 µg mL^?1 on Las‐infected ‘Valencia’ sweet orange plants [Citrus sinensis L. (Osbeck)] reduced acquisition (12 and 21% respectively) and transmission (11 and 18% respectively) of Las by feeding ACP adults compared with the controls; however, these reductions were not statistically significant. CONCLUSIONS: Pymetrozine exhibited moderate antifeedant effects by modifying the feeding behavior of ACP adults with short residual activity. The impact of pymetrozine on survival of nymphs was greater than on adults at the higher concentrations tested. Pymetrozine also reduced the acquisition and transmission of Las by feeding ACP adults up to 21 and 18%, respectively, compared with untreated controls. Copyright © 2010 Society of Chemical Industry     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).