Multiplication kinetics of Flavescence dorée phytoplasma in broad bean. Effect of phytoplasma strain and temperature

The multiplication kinetics of the Flavescence dorée phytoplasma in broad bean after inoculation by the experimental vector Euscelidius variegatus was determined. The number of phytoplasma cells, measured by quantitative real-time PCR in the aerial parts of the plants, increased exponentially over the time. After 22 to 30 days post inoculation, when symptoms appeared, bacterial growth reached a stationary phase. Whatever the time following inoculation there were no statistical differences between numbers of phytoplasma cells in plants infected by Map-FD1 (FD-CAM05) and Map-FD2 (FD92 and FD-PEY05) genotype strains. On the contrary, temperature had an influence on Flavescence dorée phytoplasma multiplication which was nearly twice as fast in broad beans incubated at 25 °C than in broad beans incubated at 20 °C. At 25 °C, plants expressed symptoms 1 week earlier. In a context of climate change, the consequences of a global warming on the Flavescence dorée epidemics are discussed.     

Spread and interaction of Pepino mosaic virus (PepMV) and Pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield

Pepino mosaic virus (PepMV) was shown to be efficiently transmitted between tomato plants grown in a closed recirculating hydroponic system. PepMV was detected in all plant parts after transmission via contaminated nutrient solution using ELISA, immunocapture RT‐PCR, RT‐PCR, electron microscopy, and by inoculation to indicator plants. Detection of PepMV in nutrient solution was only possible after concentration by ultracentrifugation followed by RT‐PCR. Roots tested positive for PepMV 1–3 weeks after inoculation, and subsequently a rapid spread from the roots into the young leaves and developing fruits was found within 1 week. PepMV was only occasionally detected in the older leaves. None of the infected plants showed any symptoms on fruits, leaves or other organs. Pre‐infection of roots of tomato cv. Hildares with Pythium aphanidermatum significantly delayed PepMV root infections. When mechanically inoculated with PepMV at the 2–4 leaf stage, yield loss was observed in all plants. However, only plants of cv. Castle Rock recorded significant yield losses when infected via contaminated nutrient solution. Yield losses induced by infection with PepMV and/or P. aphanidermatum ranged from 0·4 up to 40% depending on experimental conditions.     

Biochemical responses of coffee resistance against Meloidogyne exigua mediated by silicon

Coffea arabica cultivars Catuaí 44 and IAPAR 59, susceptible and resistant, respectively, to the root knot nematode Meloidogyne exigua, were grown in pots containing Si‐deficient soil amended with either calcium silicate (+Si) or calcium carbonate (?Si). There was an increase of 152 and 100%, respectively, in Si content of root tissue of cvs Catuaí 44 and IAPAR 59 in the +Si compared to the ?Si treatment, but no significant difference between Si treatments for calcium content. Plants, assessed 150 days after inoculation (d.a.i.) showed that the number of galls (NG) and number of eggs (NE) significantly decreased by 16·8 and 28·1% respectively, for susceptible cv. Catuaí 44 in the presence of Si, whilst both NG and NE were significantly lower for cv. IAPAR 59 compared to the susceptible cultivar regardless of Si treatments. In a separate experiment, biochemical assays were carried out 5 and 10 d.a.i. There was no significant difference between Si treatments and cultivars for concentration of total soluble phenolics. The concentration of lignin‐thioglycolic acid (LTGA) derivatives significantly increased by 11·5% in roots of nematode‐inoculated plants of susceptible cv. Catuaí supplied with Si. In roots of inoculated plants of resistant cv. IAPAR 59, the increase was 23 and 10%, respectively, for treatments with and without silicon. Peroxidase (POX), polyphenoloxidase (PPO) and phenylalanine ammonia lyase (PAL) activities significantly increased in roots of inoculated plants compared with roots of non‐inoculated plants, regardless of cultivar or Si treatment. In +Si treatments at 10 d.a.i., POX activity in roots of nematode‐inoculated plants of cvs Catuaí 44 and IAPAR 59 increased by 39·9 and 31·3%, respectively; PPO increased by 54·9 and 56·1%; and PAL activity was also higher at 26·6 and 62·9%. It was concluded that supplying Si to coffee plants increases root resistance against M. exigua by decreasing its reproductive capacity.     

The yam nematode (Scutellonema bradys), a potential threat to potato (Solanum tuberosum) production in West Africa

Potato (Solanum tuberosum) production in Africa is rapidly expanding and becoming increasingly important. As its geographical production range broadens, so does its potential to host new pests and diseases. Following the discovery that potato can be affected by Scutellonema bradys, further studies were undertaken to assess its potential pathogenicity on potato under screenhouse and field conditions, and on marketed tubers. Potato plants inoculated with S. bradys produced tubers with substantial cracking and evident tuber rot, compared with tubers from uninoculated plants. Symptoms of nematode infection on tubers included a scaly appearance, surface cracking as well as deeper tissue cracks, distortions, and darkened surface patches. In most cases these patches were related to sub‐surface rot. Nematodes were recovered from the soil, roots and tubers of inoculated plants. Eight weeks after inoculation, the reproduction factor of the nematode was greatest (2·0) at the lowest inoculation rate assessed (1000 nematodes per 2·5‐L pot) and least (0·4) at the highest inoculation rate (5000 nematodes per pot). In the screenhouse, potato tuber weights were low and mostly unaffected by nematode inoculation rate, except at 5000 nematodes per pot. In the field, non‐inoculated plants yielded over nine times more tubers than plants inoculated with 2000 S. bradys. Low densities of S. bradys were also recovered from 10 of 15 (67%) samples collected from market stalls, indicating field infection. This study confirms that potato can host and be damaged by S. bradys, raising its prospect as a likely significant biotic constraint to the crop.     

Infection of canola by secondary zoospores of Plasmodiophora brassicae produced on a nonhost

Clubroot, caused by Plasmodiophora brassicae, has two infection stages (primary and secondary). Although primary infection occurs in many plant species, secondary infection only continues to completion in susceptible hosts. As part of a larger study of clubroot pathogenesis, secondary zoospores collected from infected root hairs of canola and ryegrass were inoculated onto healthy roots of both plant species. The treatments consisted of all possible combinations of the two plant species and the two sources of inoculum. At 5 days after inoculation, levels of root hair infection were similar and in a range of 50–68% on roots in all of the treatments. Secondary infection was also observed from all of the treatments, with approximately 50% on canola and 40% on ryegrass. The proportion of secondary infection and the number of secondary plasmodia were higher in canola inoculated with zoospores from canola than in ryegrass inoculated with zoospores from ryegrass, with the other combinations intermediate. At 35 days after inoculation, typical clubs developed on 14% of the canola plants inoculated with secondary zoospores from canola, and tiny clubs developed on 16% of the canola plants inoculated with zoospores from ryegrass. Secondary infection occurred in about one-third of ryegrass plants but no clubs developed, regardless of inoculum source. These results indicate that resistance to secondary infection in ryegrass is induced during primary infection. This is the first report that secondary zoospores produced on a nonhost can infect a host and reconfirms that secondary infection can occur in a nonhost.     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

Effects of organic wastes,Glomus intraradices andPseudomonas putida on the growth of tomato and on the reproduction of the Root-knot nematodeMeloidogyne incognita

Effects of organic wastes (biosolids, horse manure, sawdust and neem leaf litter [NLL]), an arbuscular mycorrhizal (AM) fungusGlomus intraradices, and a plant growth-promoting rhizobacteriumPseudomonas putida, were studied on the growth of tomato and on the reproduction ofMeloidogvne incognita. Pseudomonas putida andG. intraradices promoted tomato growth in nematode-infected and nematode-free plants but growth promotion was higher in the infected ones. WhenP. putida andG. intraradices were applied together, the increase in tomato growth was greater than when either agent was applied alone. Of the organic wastes, NLL was better in improving tomato growth of nematode-infected plants followed by biosolids, horse manure and sawdust. Combined use of NLL withP. putida plusG. intraradices was best in improving growth of the infected plants. Root colonization byP. putida was increased more when inoculated withG. intraradices than when inoculated singly. Of the organic wastes, use of sawdust withP. putida caused a greater increase in root colonization by fluorescent pseudomonads followed by NLL, horse manure and biosolids. Nematode parasitism had an adverse effect on root colonization byP. putida. Inoculation ofP. putida and organic wastes increased the root colonization caused by the AM fungus.P. putida was better in reducing galling and nematode multiplication thanG. intraradices, whereas use of the two together was better than that of either of them alone. Among organic wastes, NLL was better in reducing galling and nematode multiplication followed by biosolids, horse manure and sawdust. Combined use of NLL withP. putida plusG. intraradices was better in reducing galling and nematode multiplication than any other treatment.     

Symptom development,in planta distribution,and transmission of <Emphasis Type="Italic">Impatiens necrotic spot virus</Emphasis> in gentian: evidence for survival in roots and winter buds

Impatiens necrotic spot virus (INSV) causes serious damage to gentian (Gentiana spp.). Symptom development, in planta distribution, and transmission of INSV were studied after mechanical inoculation of gentian plants and propagation of shoot cuttings from infected stock plants. When young gentian plants at the 2nd-leaf stage were inoculated with INSV, plants developed systemic symptoms that were restricted to a few upper leaves. However, older plants at the 6th-leaf stage did not develop systemic symptoms. After plant inoculation, INSV was detected using DAS-ELISA in symptomatic upper leaves, and rootlets and winter buds, but not in asymptomatic leaves. When asymptomatic shoot cuttings from infected stock plants were vegetatively propagated in a thrips-free glasshouse, 44.4% of those obtained from the apical shoot and 20.6% of those obtained from the middle section of the plant developed systemic symptoms. These results indicated that when gentian plants were infected with INSV, the virus was preferentially transported from the infected leaves to the root and winter buds. However, even asymptomatic shoot cuttings may develop systemic symptoms when obtained from infected stock plants. Therefore, vegetative propagation from infected stock plants can be a source of INSV infection.     

Transmission and detection of toria [<Emphasis Type="Italic">Brassica rapa</Emphasis> L. subsp. <Emphasis Type="Italic">dichotoma</Emphasis> (Roxb.)] phyllody phytoplasma and identification of a potential vector

Phyllody disease associated with 16SrIX phytoplasma was observed in the range of 4.1–11% in 10 different lines of toria [Brassica rapa L. subsp. dichotoma (Roxb.)] in experimental fields of the Indian Agricultural Research Institute, New Delhi, India during 2008 and 2009. The toria phyllody (TP) phytoplasma was detected in all the symptomatic and 13.3% of asymptomatic toria plants by nested PCR. The phytoplasma was detected in midrib, flower part, siliquae, stem, and root of infected plants as well as seeds. TP was transmitted by grafting and by dodder to toria and nine other rapeseed/mustard species as confirmed by nested PCR. However, symptoms of phytoplasma infection were induced only in toria, yellow sarson [Brassica rapa L. subsp. trilocularis (Roxb.)], brown sarson [Brassica rapa L. subsp. sarson (Prain)], rapeseed (B. napus subsp. oleifera), and rocket or taramira (Eruca sativa) but not in mustard (B. juncea), black mustard (B. nigra), Ethiopian mustard (B. carinata), B. tournefortii and white mustard (Sinapis alba). Transmission of TP phytoplasma to periwinkle (Catharanthus roseus) was successful only through dodder, but no transmission to tomato (Lycopersicon esculentum) or brinjal (Solanum melongena) was found. TP phytoplasma was detected in Laodelpax striatellus, an abundant planthopper in toria fields, which indicates that this planthopper may be a potential vector for TP phytoplasma.     

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.     

In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.     

Development of Oculimacula yallundae and O. acuformis (eyespot) on leaf sheaths of winter wheat in the UK in relation to thermal time

In a controlled environment (15/10°C) (day/night) container experiment on winter wheat (cv. Avalon), eyespot incidence (percentage of plants affected) and number of leaf sheaths penetrated after 6 weeks increased with inoculum concentration (10²−10⁶ conidia mL⁻¹) of Oculimacula yallundae (OY) or Oculimacula acuformis (OA), but there was no difference between the two species. In an outdoor container experiment, seedlings inoculated with OY 2 weeks after sowing had a greater incidence of eyespot than those inoculated with OA, when assessed 7 weeks after inoculation. Seedlings inoculated with OA at 10 or 20 weeks after sowing developed more severe eyespot by maturity than those inoculated with OY. In an experiment at 15/10°C with seedlings inoculated with OY + OA 2 weeks after sowing, more leaf sheaths were penetrated by OY (3·0 per plant) than OA (2·3 per plant) 6 weeks after inoculation. Field experiments with winter wheat consistently showed leaf sheath production, leaf sheath death, and number of leaf sheaths infected or penetrated by OA or OY were related linearly to thermal time (°C days) after sowing. Depending on cultivar, season and sample, a new leaf sheath was produced in 116–216°C days; a leaf sheath died in 221–350°C days; and infection of a new leaf sheath occurred in 129–389°C days. The mean number of living leaf sheaths infected differed between samples, cultivars and seasons for both OY and OA. Regression analysis of the 1985/86 data suggested that OY progressed more rapidly than OA through the leaf sheaths, and that both the pathogens progressed more rapidly than the rate of leaf sheath death, but more slowly than the rate at which leaf sheaths were produced. It also suggested that OA progressed more slowly than the rate at which leaf sheaths died in 1987/88, but OY did not.     

Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean C_t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean C_t value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C_t value in mature leaves = 33·6).     

Infection of potato by Rhizoctonia solani: effect of anastomosis group

Glasshouse and field experiments showed that the pathogenicity and disease type on potato varied between different anastomosis groups (AGs) of Rhizoctonia solani. For example, severe stem and stolon disease developed in plants inoculated with a single isolate of AG3PT and AG5. Severe root disease was observed with single isolates of AG8 and to a lesser extent AG3PT, but rarely with single isolates of the other AGs tested. In both field and glasshouse experiments the AG2‐1 isolate (X81) produced only small lesions (<5 mm). However, this was not representative of two other AG2‐1 isolates. When AG2‐1 isolates of the three different rDNA IGS1 types were tested in a glasshouse trial, one caused more severe stem and stolon infection than AG3PT. In the field experiment, the yield of tubers, by weight, was significantly less (P < 0·05) in all inoculated plants than for uninoculated (control) plants. Yield losses were greatest and tuber numbers smallest in plots inoculated with an AG8 isolate, suggesting that root infection is important in determining quantitative yield loss. The incidence of black scurf was greatest in the progeny tubers in plots inoculated with AG3PT (83·9%), whereas only very small amounts of black scurf developed on tubers from plants infected with AG2‐1 (510 bp) or AG5 isolates. This is supported by laboratory tests, where isolates of AG3PT produced significantly more sclerotia on potato dextrose agar than isolates of AGs 2‐1, 4, 5 and 8.     

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.     

Potential role of endogeic earthworm <Emphasis Type="Italic">Pontoscolex corethrurus</Emphasis> in remediating banana blood disease: a preliminary observation

Blood disease is a destructive bacterial infection that causes severe yield loss to the banana industry. Ideally, an environmental friendly yet practical approach is necessitated in the search for effective treatment against the disease. Endogeic earthworms are soil biota that help in improving soil physico-chemical and biological properties and thuspromote plant health. The present study assessed the effect of Pontoscolex corethrurus on banana infected by blood disease. The effect of earthworms was evaluated through observations on stem and root morphology, anatomy and total phenolic contents (TPC). P. corethrurus was inoculated into polybags planted with banana plantlets; inoculation of blood disease bacterium (BDB) into the roots of the plantlets was done using a drenching method. Stem and root sections of the plantlets were preserved in FAA for histological study upon harvest. The remaining sections were freeze-dried for TPC analysis. Root sections of plantlets infected with BDB showed lower root biomass compared to the control and earthworm-inoculated plantlets. Observations under microscope also showed that tissue necrosis at the vascular bundles of infected roots were more severe compared to the earthworm-inoculated and unaffected plantlets. Plantlets inoculated with earthworms had the highest root TPC, followed by the unaffected plantlets and plantlets infected with BDB. Although infected plantlets with earthworm inoculation showed disease symptoms, the disease severity was slightly less. The results suggested the potential role of P. corethrurus in improving plant health for disease management and sustainable agriculture.     

Movement of <Emphasis Type="Italic">Cucumber Mosaic Virus</Emphasis> Is Restricted at the Interface between Mesophyll and Phloem Pathway in <Emphasis Type="Italic">Cucumis figarei</Emphasis>

Viral movement in the leaf tissues of a resistant host, Cucumis figarei, inoculated with the pepo strain of Cucumber mosaic virus (CMV) and incubated at 24°C or 36°C was investigated by fluorescence in situ hybridization (FISH), leaf-press blotting, tissue printing and immunogold-silver staining techniques. Observation by FISH revealed that at 24°C most infection sites with CMV at 0.01 mg/ml or 0.1 mg/ml were limited to a single cell during the incubation period, that the number of infection sites increased from 24hpi (hours post inoculation) to 80 hpi in the leaves inoculated with CMV at 0.5 mg/ml, and that the size as well as the number of infection sites rapidly increased with time in the leaves inoculated with CMV at 2.0 mg/ml. These results suggested that one factor for the resistance of C. figarei at 24°C might be an inhibition of viral movement in and out of the infection sites. Leaf-press blotting and tissue blotting indicated that CMV remained in the infection sites at 24°C, whereas it spread from the inoculated leaves to other parts of the plants through vascular systems at 36°C. Immunogold-silver staining demonstrated that at 24°C CMV infected bundle sheath (BS) cells in minor veins, whereas at 36°C it invaded not only BS cells, but also phloem parenchyma (PP)/ companion cell (CC) or PP/intermediary cell (IC) complexes in minor veins in the regions with chlorotic symptoms. These results indicated that at 24°C CMV had difficulty in passing through the interface between BS and PP/CC or PP/ IC complexes and that viral entry from mesophyll to the phloem pathway was inhibited in the inoculated leaves. Received 26 August 1999/ Accepted in revised form 14 December 1999     

Infection and colonization of strawberry by <Emphasis Type="Italic">Gnomonia fragariae</Emphasis> strain expressing green fluorescent protein

Gnomonia fragariae is a poorly studied ascomycete, which was recently demonstrated to be a cause of severe root rot and petiole blight of strawberry. The pathogen was genetically transformed with the GFP as a vital marker and hygromycin resistance gene. Several stable transformants were obtained, which did not differ in their phenotype from the wild type isolate. Using one of the GFP-tagged isolates the infection process and colonization of roots and petioles of host plant by the pathogen were studied. Fluorescence microscopy examinations of the inoculated plants at different time points showed that plant infection occurs 24 h after inoculation and intensively continues during first 3 days. The specific penetration sites on epidermal cells and preferences in colonization for certain root and petiole tissues were observed. The pathogen intensively colonized and destroyed cortex of roots and petioles and spread rapidly longitudinally within intercellular spaces. The petioles were colonized by the hyphae, which grew mostly in the intracellular spaces of the cortical cells while in the roots the intracellular growth of hyphae occurred only in the later stages of infection. The fungus was also capable to infect the vascular tissues of petioles although these were not the primary tissues colonized by the pathogen. The mature ascomata were formed on the infected petiole bases several weeks after the inoculation. This study presents a genetic transformation method for Gnomonia fragariae and it demonstrates details on infection process and colonization of root, crown and petiole tissues of strawberry by the pathogen.