Chocolate Spot Research Articles

Study on occurrence and importance of faba bean diseases with special consideration to the newly emerging faba bean gall in Tigray, Ethiopia

Faba bean is one of the most important crops in Ethiopia. The production of the crop is, however, constrained by several disease infections including fungal diseases. A survey was conducted in 2013 to assess the occurrence and importance diseases affecting faba bean in south Tigray. The observed diseases were gall, chocolate spot, ascochyta blight, alternaria leaf spot, black root rot and rust in order of their prevalence. The overall mean incidences of 66, 45.5, 45.9, 28.9, 5.7, 1.1, and 0.4% and with severities of 64.4, 47.5, 15.3, 7.7, 11.8 and 0.2%, respectively. Faba bean gall was the most devastating newly identified disease in Tigray. The severity of the disease ranged from 30% in Emba-Alaje to 100% in Ofla, Enda-Mekoni and Raya-Alamata districts indicating the seriousness of the disease in the area. In addition, improved varieties were relatively tolerant to most identified diseases as compared to the local cultivars except for the new “gall” disease wipes-out fields without any tolerance. Hence, it is very important to use integrated management tactics and risk forecasting that operate on different aspects of the disease etiology. Key words: Disease, faba bean, gall.

Wpływ dawki startowej azotu oraz dokarmiania dolistnego na wskaźnik LAI oraz porażenie przez patogeny grzybowe dwóch morfotypów bobiku

This paper describes the influence of initial dose of nitrogen and foliar nutrition on LAI (leaf area index) index and on infection of fungal pathogens of two morphotypes of faba beans. Occurrence of ascochyta blight (Ascochyta fabae), chocolate spots (Botrytis fabae) and faba bean rust (Uromyces fabae) was analysed. In the years 2011–2013 strict field tests were carried at the Experimental Station of Variety Evaluation in Przecław. The following factors were analysed: initial nitrogen dose (25 kg/ha), foliar nutrition (Basfoliar 6-12-6) and Granit and Amulet cultivars. The initial dose of nitrogen considerably increased LAI index in comparison with the control but the foliar nutrition did not influence LAI index as compared with the control. Amulet cultivar was better than Granit cultivar as far as tested parameters were concerned. According to the obtained results fertilization with the initial dose of nitrogen and foliar nutrition did not influence the infection severity of diseases caused by the examined fungal pathogens in respect to the control. On the other hand the cultivars were sensitive to the studied diseases at a different level and it varied in individual years. Granit cultivar as compared with Amulet was infected more by ascochyta blight and chocolate spots and less by faba bean rust. W pracy określono wpływ dawki startowej azotu oraz dokarmiania dolistnego na wskaźnik LAI (leaf area index) oraz porażenie przez grzyby chorobotwórcze dwóch morfotypów bobiku. Ocenie poddano występowanie askochytozy bobiku (Ascochyta fabae), czekoladowej plamistości bobiku (Botrytis fabae) i rdzy bobiku (Uromyces fabae). Ścisłe doświadczenie polowe przeprowadzono w latach 2011–2013 w Stacji Doświadczalnej Oceny Odmian w Przecławiu. Badanymi czynnikami były: dawka startowa azotu (25 kg/ha), dokarmianie dolistne (Basfoliar 6-12-6) oraz odmiany Granit i Amulet. Zastosowana dawka startowa azotu istotnie zwiększyła, natomiast dokarmianie dolistne nie zróżnicowało wskaźnika LAI w porównaniu do obiektu kontrolnego. Odmiana Amulet badanym parametrem przewyższyła odmianę Granit. Uzyskane wyniki badań nie wykazały istotnego wpływu nawożenia startowego azotem, jak i dolistnego dokarmiania na stopień porażenia przez badane patogeny grzybowe w odniesieniu do roślin kontrolnych. Stwierdzono odmianową podatność na oceniane choroby oraz zmienność ich nasilenia w poszczególnych latach. Odmiana Granit w porównaniu do odmiany Amulet była silniej porażona przez A. fabae i B. fabae, a w mniejszym stopniu przez U. fabae.

Antifungal properties of an actinomycin D-producing strain, Streptomyces sp. IA1, isolated from a Saharan soil.

An actinomycete strain named IA1, which produced an antimicrobial compound, was isolated from a Saharan soil in In Amenas, Algeria. The study of the 16S rDNA sequence of this strain permitted to relate it to Streptomyces mutabilis NBRC 12800(T) (99.93% of similarity). Strain IA1 exhibited strong activity against a wide range of plant pathogenic fungi. One bioactive compound produced in large amounts (46.7 mg L(-1) day(-1) ), named YA, was isolated and purified by TLC and reverse phase HPLC. The structure elucidation of the pure substance, using combined data from UV visible, NMR spectra, and mass spectrometry, permitted to identify it as actinomycin D, and was thus found for the first time in S. mutabilis related species. The biocontrol abilities of the strain IA1 and compound YA were evaluated through two diseases, i.e., chocolate spot of field bean and Fusarium wilt of flax. The occurrence of the two fungal diseases was effectively reduced. The reduction of chocolate spot disease symptoms reached 80 and 91.7% with IA1 and YA seedlings pretreatments, respectively. Soil pretreatment with IA1 or YA also allowed to reduce Fusarium wilt disease impact by almost 60%.

Specific detection of three Torradovirus species with digoxigen-labeled probes

Background Torradoviruses are an emerging group of picorna-like plant virus from the family Secoviridae that infect tomato and other Solanaceae species. The genus Torradovirus include four species: Tomato torrado virus (ToTV), first found in Europe, and afterward in Central America and Australia; Tomato apex necrosis virus (ToANV), present in Mexico; Tomato chocolate spot virus (ToChSV) and Tomato chocolate virus (ToChV), both found in Guatemala. The symptoms caused by these viruses include chlorotic regions on the leaves that may develop to necrotic spots and holes, while the fruits show necrotic lines and frequently cracks on the surface, reducing yield and quality [1]. Even though these viruses have not yet been found in Brazil, the sudden spread of ToTV from Europe to Australia and Central America emphasize the necessity of effective methods to detect them. For that reason, we have developed digoxigenin-labelled RNA probes for the detection of three torradoviruses, ToANV, ToChSV and ToTV, through hybridization.

Efficiency of Effective Microorganisms (EM) to Induce Resistance against Chocolate Spot Disease and Enhance Productivity of Faba Bean under Reclaimed Soil Conditions

Efficiency of Effective Microorganisms (EM) to Induce Resistance against Chocolate Spot Disease and Enhance Productivity of Faba Bean under Reclaimed Soil Conditions

Control of Chocolate Spot Disease by Non Traditional Methods on Faba Bean Plants

Control of Chocolate Spot Disease by Non Traditional Methods on Faba Bean Plants

Faba Bean Gall; a New Threat for Faba Bean (Vicia faba) Production in Ethiopia

Faba bean ranks first in its production volume and cultivated land among pulse crops cultivated in Ethiopia and it is valuable as the cheap source of protein in most Ethiopian diet. It also plays a significant role in soil fertility restoration and in export market. Production of the crop is, however, constrained by several pests. The objective of this study was to find out the distribution and intensity of newly occurred epidemic faba bean gall disease and other diseases affecting faba bean in the major growing areas of central and northern part of Ethiopia. The survey was made in 2013 main cropping season (in September) along the main roads and accessible routes in each surveyed district at every 5-10 km intervals as per faba bean fields available. Five samples were taken in each faba bean field by moving “X” fashion. The mean prevalence of faba bean gall, Ascochyta blight, Chocolate spot and rust were 48.5%, 63.6%, 94.6% and 1.9%, respectively. The mean incidence of the formal all diseases were 15.4%, 30.3%, 42.4% and 0.1% in their previous order. Based on severity scale, mean disease severity of Ascochyta blight and chocolate spot were 1.9 and 1.5, respectively. Mean severity of faba bean gall and faba bean rust were 6.4% and 0.1% were recorded in their order. The disease was more sever in Amhara region with mean disease severity of 22.2% followed by Tigray and Oromiya region which had 11.3% and 7.8% severity value, respectively. The newly observed Faba bean gall disease was found the most devastating and widely disseminated in the area within a few years since its occurrence. The production of the crop is highly challenged and farmers are frustrated by the nature of the disease. Farmers witnessed the appearance of the disease three years ago in 2011 in Oromiya. They observed fast dissemination and increasing coverage of the pathogen in time and space. In fact the disease is epidemic and more serious from the record and the information from Faba bean growers in the surveyed areas. The epidemic conditions of the disease have significant implication on the production of Faba bean and on the country’s economy. This survey information helps to consider the disease as serious pest in Ethiopia and in development of management options. In the future, ecology and biology of the causative agent, yield loss due to the diseases, breeding for disease resistance/tolerance and management strategies should get attention.

Effect of Volatile and Non-Volatile Compounds of Trichoderma spp. on Botrytis Fabae the Causative Agent of Faba Bean Chocolate Spot

Antagonistic fungi naturally occurring on faba bean leaf surface were isolated and evaluated for their activity as bioagents for Botrytis fabae the causative agent of chocolate spot disease. Thirty isolates were purified and identified as 26 isolates of Trichoderma species (Trichoderma album, T. aureoviride , T. hamatum, T. harzianum and T. viride) and 4 isolates belonging to the genera of Cladosporium, Gliocladium, Epicoccum and Paecilomyces. The inhibitory effect of these isolates was assessed in vitro against the growth of B. fabae, which decreased it's mycelial growth on PDA plates. The inhibitory effect of Trichoderma spp. ranged between 51.11 - 77.78%. In addition, T. album (Isolate 2) gave the highest inhibition followed by T. harzianum (Isolate 6). Furthermore, under greenhouse conditions spraying of faba bean plants with any of Trichoderma spp. and Bio- Zeid as a biofungicide, 24 h before inoculation with B. fabae significantly reduced the severity of the disease after 14 days in the range of 3.0 - 4% compared with the control (8.7%). T. album (Isolate 2) was the highest antagonistic isolate (3.0%) followed by T. harzianum (Isolate 6) then T. hamatum (Isolate 6) and T. viride (Isolate 2), being 3.24, 3.30 and 3.40%, respectively. Volatile and non-volatile compounds produced by T. album (Isolate 2) exhibited the highest inhibition to the mycelial growth of B. fabae followed by T. harzianum (Isolate 6).

English

Pseudomonas fluorescens isolates possess a variety of promising properties of antifungal activity of secondary metabolites which make it as a biocontrol agent. In the present study, 12 isolates of P. fluorescens were isolated from rhizospheric soil of faba bean crop evaluated for their antagonistic activity against chocolate spot disease (Botrytis fabae) of faba bean. P. fluorescens 10 (Pf 10) (88.1%) showed high antagonistic activity against B. fabae. All isolate of P. fluorescens were successfully employed in controlling chocolate spot disease of faba bean due to their antifungal metabolites. The antifungal compounds were extracted from all P. fluorescens isolates with equal volume of ethyl acetate, hexane and methanol. The antifungal compounds extracted with ethyl acetate, hexane and methanol from P. fluorescens 3 (Pf 3), P. fluorescens 8 (Pf 8), and P. fluorescens 3 (Pf 3), isolates at 0.1% concentration completely inhibited the mycelial growth of the pathogen respectively. Bio-primed faba bean seeds with isolates of P. fluorescens 9 (Pf 9) and P. fluorescens 10 (Pf 10) evaluated against B. fabae in vivo (pot culture) indicated the inhibitory effects to the pathogen and also showed the inducing properties to enhance the immune system of crop. Therefore, it can be concluded that the use of P. fluorescens 9 (Pf 9) and P. fluorescens 10 (Pf 10) of isolates could inhibit the mycelial growth and reduced the disease incidence, severity and infection processes of B. fabae and simultaneously increase the plant growth performance and yield of faba bean. These isolates can be used as potential biocontrol agents against B. fabae and also used as biofertilizers for the production of faba bean. Key words: Antifungal, Biocontrol, Botrytis fabae, Faba bean, Pseudomonas fluorescens.

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 107 and 2 × 107 spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.

Pseudomonas fluorescens isolates as an inducer of physiological activities of faba bean (Vicia faba)

Plant protection is an important area which needs attention since most of the hazardous inputs added into the agricultural system are in the form of plant protection chemicals. Production of the crop is, however, constrained by fungal plant pathogens infections. The present study was also carried out for estimation of total phenols and flavonoids present in fresh faba bean leaf extract with ethanol. Applying the Pseudomonas fluorescens 9 and 10 by bio-primed seed of faba bean treatment enhanced the accumulation of total phenols and flavonoids compared to untreated infected and uninfected untreated faba bean. Bio-priming faba bean seeds with P f9 and P f10 were increased the faba bean total flavonoids in NC 58, Moti and ILB 938 varieties with range 450.58 to 770.19 mg/g compared negative and positive control. The maximum amount of total phenol was recorded 152.25 mg/g in Moti variety with P f9 isolate and 123.70 mg/g was recorded in ILB 938 variety with P f10 isolates. On another hand, all isolates of P. fluorescens were tested for certain biochemical production. Consequently all isolate are positive for all tests. Therefore, from this study it is possible to conclude that the use of P. fluorescens 9 and 10 isolates could reduce the disease incidence and severity of caused by chocolate spot (Botrytis fabae) and simultaneously increased the plant growth promoting and yield performance of faba bean. These isolates can be used as potential biofertilizers, inducer of ant-fungal compounds and also as biocontrol agent against Botrytis fabae. So all isolates of P. fluorescens can be used as plant growth promoting producing bacteria for increasing the yield production and quality of faba bean. Key words: Botrytis fabae, faba bean, flavonoids, phenols, Pseudomonas fluorescens.

In vivo Assay for Antagonistic Potential of Fungal Isolates against Faba bean ( Vicia faba L. ) Chocolate Spot ( Botrytis fabae Sard. )

Due to its high protein content, faba bean (Vicia fabae L.) leaves harbor many microorganisms besides Botrytis fabae. The objective of this study is to explore fungal isolates residing on faba bean leaves and evaluate their antagonistic potential against B. fabae. For this matter, 236 leaf samples were collected from different districts of West Hararghe and Bale zones. Out of which 72 fungal species were isolated and evaluated for their in vivo biocontrol potential against chocolate spot (B. fabae Sard.). The in vivo assay was conducted in two stages where detached leaf test and intact leaf test was involved. Significant difference (p<0.05) resulted among fungal isolates to affect incubation period (IP) and disease severity (DS) on local and Shalo cultivars on detached leaf. Isolates GO2-3, GB6-3, S16-2, A12-1 and 52-BT resulted incubation period of 3.7 - 4.7 days, where it was 2 days on untreated control of both cultivars. Lower disease severity was recorded from the leaf treated by GO2-3 and S16-2 on local and GO3-2 on Shalo based on 1-4 rating scale. On intact plant, significant difference (p<0.05) among fungal isolates was resulted to affect IP, diseases incidence (DI) and DS. Higher IP was recorded from isolates GB6-3 (3.3), S16-2 (3), and GO3-2 (3 days) on local. GO3-2 showed better reduction (66.7 %) of chocolate spot incidence on Obse compared to the control (100%). Isolates S16-2, GO3-2 and GB6-3 resulted lower disease severity (percent severity index) of 35.6-51.1% as compared to control (73.3-84.4%) on the three cultivars.

PLANT NUTRIENTS AS CONTROLLING OF CHOCOLATE SPOT, RUST DISEASES AND IMPROVING THE PERFORMANCE OF FABA BEAN

Fungal disease i.e.,chocolate spot (Botrytis fabae Sard.)and rust (Uromyces vicia fabae (Pers.) Schart) are the most destructive diseases of faba bean and causes considerable dramatic damage losses on faba bean yield worldwide Therefore, the use of plant nutrients (leaves fertilizers) i.e., Ascobein(As), Citreen(Ci) and Potassen (Po) to both diseases (chocolate spot and rust) in Vicia fabae ,gave evidence for controlling these diseases. Results showed that ,spraying faba bean plants with As+Ci+Po significantly decreased chocolate spot disease with 3.5% and 3.81% while ,rust was by decreased 5.2% and 5.77% for the two growing seasons 2010/2011 and 2011/2012. Total chlorophyll reached to the maximum values when using Potassen (1.933mg/g f.w.) while ,As+Ci+Po as combination revealed the most superior effect on total phenolics compounds .All treatments were found to be most effective in increasing growth characters and yield of faba bean.

Improvement of faba bean (Vicia faba L.) yield and quality through biotechnological approach: A review

Faba bean ( Vicia faba L.), an old-world grain legume, is grown approximately in 3 m/ha area world-wide from temperate, tropical to hot arid conditions. It is such a wonderful legume that it can excel even under adverse soil conditions; acidic or saline alkaline (pH 4.5 to 9.0). In favourable conditions, it gives very high yields, but low yield may result from biotic and abiotic stress. In India, it is still treated as minor legume. Genetic transformation based on Agrobacteria is possible. Several random amplified polymorphic DNA (RAPD) markers linked to a gene determining hypersensitive resistance to race 1 of the rust ( Uromyces viciae-fabae ) have been reported. Molecular breeding for resistance to broomrape, Ascochyta blight, rust, and chocolate spot have been obtained. The use of marker assisted selection (MAS) can complement conventional breeding by speeding up the selection of desirable traits and increasing selection efficiency. Recently, markers linked to a gene controlling growth habit or to select against traits affecting the nutritional value of seeds have also been reported. Lack of suitable cultivar can be easily overcome by application of modern tools and techniques. Several in-vitro techniques would be very useful for faba bean breeding. New techniques such as protoplast fusion, regeneration, and embryo-rescue assisted interspecific crossing could probably be introduced to V. faba L. to improve yield and quality. This review work examines the role of various techniques with reference to faba bean improvement. Key words: Vicia faba , faba bean, grain quality, resistance breeding, nitrogen fixation, zero tannin.

Biochemical screening of chocolate spot disease on faba bean caused by Botrytis fabae

In this work, three faba bean cultivars were studied. The cultivar Giza 429 was the most susceptible cultivar to chocolate spot disease, followed by Giza 40, which was moderately susceptible; cultivar Giza 461 was found to be resistant. Four fungal species associated with diseased leaves were isolated from naturally infected samples and identified asBotrytis fabae, Alternaria alternata and Stemphylium sp. The most frequently isolated fungus was B. fabae, with an average isolation frequency of 51.03%. Of the B. fabaeisolates, six were pathogenic in Giza 429 and Giza 40, and the symptoms of chocolate spot appeared three days after inoculation: B. fabae isolate No. 1 was the most virulent, whereas isolate No. 6 was the least virulent. The effect of B. fabae infection in the twovarieties caused biochemical changes in the content of phenolics, oxidative enzymes,phytoalexins and free amino acids. HPLC analysis showed that the resistant cultivar Giza 461 demonstrated significantly higher amounts of oxidative enzymes, free, conjugated and total phenols and phytoalexins (wyerone acid, wyerone and wyerone epoxide) compared with the highly susceptible cultivar Giza 429. The results of this study may improve our understanding of the biochemical basis of resistance to B. fabae in the faba bean. Key words: Faba bean, Botrytis fabae, chocolate spot disease, phenolics, oxidative enzymes and phytoalexins, fractionation of amino acids.

PRODUCTIVITY AND CHOCOLATE SPOT AND RUST DISEASES OF SOME FABA BEAN CULTIVARS UNDER DIFFERENT SOWING DATES AND SALICYLIC ACID CONCENTRATES

Two experiments were conducted at the experimental Farm at Tag El-Ezz, Dakahlia, Egypt, during 2008/2009 and 2009/2010 to evaluate the productivity, chocolate spot and rust diseases for five faba bean cultivars under two sowing dates and three salicylic acid concentrations. Sowing date November 15th gave the highest values of morphological and yield characters while, gave the lowest values of chocolate spot and rust compared with October 15th sowing date in both seasons. Seed yield/fed. recorded 8.17 and 7.49 ardab**/fed*. for sowing date November 15th and 6.17 and 6.15 ardab/fed. for the sowing date October 15th in both seasons, respectively. Giza 716 produced the highest seed yield/fed. in the second season, but no significant differences were observed between faba bean cultivars in the first season. The maximum reduction in severity of chocolate spot and rust diseases accrued under the application of salicylic acid (SA) at 150 mg/L. at the two sowing dates in both seasons. Whereas, led to marked improvement in morphological characteristics and yield. The interaction between sowing date and faba bean cultivars led to significant increase in plant height, straw yield, seed weight/plant and seed yield in both seasons. The maximum reduction of chocolate spot and rust disease severity was recorded with Giza 716 in November 15th planted under application of SA at 150 mg/L. The correlation coefficient cleared significant positive correlation between seed yield and each of individual studied characters. Multiple regression analysis indicated that the relative contribution for all characters gave 93.06% from the total variations of yield. Stepwise regression revealed that three out of six variables significantly affected seed yield/plant, these variables were seed weight, number of branches and number of pods per plant. The present investigation recommends to planting Giza 716 cultivar in November 15th with spraying of SA (150mg/l.) after 50 and 70 days from sowing to decrease the severity of chocolate spot and rust diseases and improve faba bean productivity.

Two generic PCR primer sets for the detection of members of the genus Torradovirus

Two degenerate primer pairs were designed for the universal detection of members of the genus Torradovirus. Primer pair Torrado-1F/Torrado-1R was designed based on the RNA-dependent RNA polymerase region located in RNA1 and primer pair Torrado-2F/Torrado-2R based on a region overlapping the two first coat proteins Vp35 and Vp26 in RNA2. The primers were used in two-step and one-step RT-PCR protocols. Both primer pairs were able to detect 14 out of 15 isolates belonging to the two torradovirus species Tomato torrado virus (ToTV) and Tomato marchitez virus (ToMarV) and the two tentative species Tomato chocolate spot virus (ToChSV) and Tomato chocolàte virus (ToChV). Due to poor sample quality, one isolate of ToTV was detected with primer pair Torrado-2F/Torrado-2R and not with primer pair Torrado-1F/Torrado-1R, suggesting that the latter primer pair was less sensitive. Nevertheless, both primer pairs proved to be suitable for the universal RT-PCR detection of torradoviruses and can be deployed for the detection of all currently known torradoviruses and possibly for the detection of new members of this group.

Influence of Essential Oils of some Medicinal and Aromatic Plants on Growth and Control of Botrytis fabae the Causal of Faba Bean Chocolate Spot

Influence of Essential Oils of some Medicinal and Aromatic Plants on Growth and Control of Botrytis fabae the Causal of Faba Bean Chocolate Spot

Pathogenic and genetic diversity of Botrytis fabae Sand. isolates from faba bean fields in different agro-ecological zones of Northern Ethiopia

The identity and genetic diversity of the causal agent of chocolate spot (Botrytis cinerea or Botrytis fabae) in Ethiopia is still poorly defined and this hinders efforts aimed at integrated management of the disease, especially breeding for resistance. Chocolate spot disease epidemics are more severe in the moist, humid western districts of northern Ethiopia (NW) compared to the sub-humid eastern districts (NE) but knowledge of pathogen identity and genetic diversity is needed to facilitate epidemiological studies. Of a total of 120 Botrytis isolates examined, all were found to be B. fabae and none fitted the morphological description of B.cinerea. In pathogenicity tests, representative isolates caused typical chocolate spot symptoms and were re-isolated from infected leaves, indicating that B. fabae is the causal agent of chocolate spot in Ethiopia. Marked differences were found among the isolates in colony morphology and growth rate but the morphology of isolates was unrelated to their pathogenicity. A preliminary virulence (=severity of disease) test with 76 isolates revealed differences in virulence of the B. fabae isolates, with NE isolates being generally more virulent than the NW isolates. Genetic analysis based on 129 amplified fragment length polymorphism (AFLP) markers showed that genetic differentiation among pathogen populations was low (GST = 0.02 to 0.03; θ = 0.02, p = 0.05), indicating limited geographic delimitation and significant gene flow. Total gene diversity (HT = 0.22) was mostly attributable to diversity within populations (HS = 0.21). Thus, only 4.5% of the total variability was attributable to frequency differences between agroecological zones. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from NW and NE, and from the moist and subhumid agro-ecological zones (GD = 0.99). However, cluster analysis showed high similarity among many isolates (>75% similarity index), suggesting that such isolates have a familial structure or are clonally related. The phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on virulence or agro-ecological zone. The implications of these findings for chocolate spot resistance breeding in Ethiopia are discussed.

First Report of Tomato torrado virus Infecting Tomato in Colombia.

Tomato (Solanum lycopersicum L.) plants grown in plastic greenhouses near Villa de Leyva, northeast of Bogota, Colombia showed necrotic spots on the leaves in September 2008. Initial symptoms were necrosis beginning at the base of leaflets that were surrounded by yellow areas. These symptoms resembled those described for Tomato torrado virus (ToTV; family Secoviridae, genus Torradovirus), which was first found in Spain (2). Other (tentative) members of the genus Torradovirus, Tomato marchitez virus (ToMarV), Tomato chocolate spot virus (ToChSV), and Tomato chocolàte virus (ToChV) (3) induce similar symptoms on tomato plants. One sample, coded T418, was stored in the freezer and brought to our lab in 2011. Serological tests (double-antibody sandwich-ELISA) using polyclonal antibodies (Prime Diagnostics, Wageningen, The Netherlands) on leaf extracts showed the absence of Pepino mosaic virus (PepMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY). Leaf extracts were mechanically inoculated onto the indicator plants Physalis floridana, Nicotiana hesperis '67A', and N. occidentalis 'P1' (six plants in total) and were kept in a greenhouse at 20°C with 16 h of light. Necrotic symptoms appeared 4 to 5 days postinoculation and resembled those described for ToTV (2). Two dip preparations of systemically infected P. floridana and N. occidentalis leaves were examined by electron microscopy, which revealed the presence of spherical virus particles of approximately 30 nm. To confirm the presence of ToTV, total RNA was extracted from the original leaf material and an inoculated P. floridana and N. occidentalis plant using the Qiagen Plant Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. ToTV-specific primer sets ToTV-Dp33F/ToTV-Dp20R (5'-TGCTCAATGTTGGAAACCCC-3'/5'-AGCCCTTCATAGGCTAGCC-3', amplifying a fragment of the RNA1 polyprotein with an expected size of 751 bp) and ToTV-Dp1F/ToTV-Dp2R (5'-ACAAGAGGAGCTTGACGAGG-3'/5'-AAAGGTAGTGTAATGGTCGG-3', amplifying a fragment on the RNA2 movement protein region with an expected size of 568 bp) were used to amplify the indicated regions in a reverse transcription (RT)-PCR using the One-Step Access RT-PCR system (Promega, Madison, WI). Amplicons of the predicted size were obtained in all tested materials. The PCR products were purified with the Qiaquick PCR Purification Kit (Qiagen) and sequenced directly. BLAST analyses of the obtained sequences (GenBank Accession Nos. JQ314230 and JQ314229) confirmed the identity of isolate T418 as ToTV, with 99% identity to isolate PRI-ToTV0301 in both fragments (GenBank Accession Nos. DQ388879 and DQ388880 for RNA1 and RNA 2, respectively). To our knowledge, this is the first report of ToTV in Colombia, and interestingly, since ToTV has been found only in Europe and Australia (1) so far, this is the first report of ToTV on the American continent.