Variation in Aggressiveness Components in the Hemileia vastatrix Population in Brazil

The Pucciniomycete fungus Hemileia vastatrix causes leaf rust on coffee trees. The pathogen is responsible for considerable yield losses in susceptible coffee cultivars if appropriate management strategies are not implemented. Rapid spread and epidemics of rust fungi are usually associated with the emergence of new races of the pathogen that overcome resistance or with the emergence of more aggressive populations of the pathogen. In Brazil, coffee production is dominated by susceptible cultivars of Coffea arabica and Coffea canephora. We assessed aggressiveness in 46 populations of H. vastatrix from Minas Gerais and Espírito Santo, two of the most important coffee‐producing states in Brazil. We observed a significant difference in the incubation period between the populations from Minas Gerais and Espírito Santo when 183 single‐pustule isolates were inoculated onto Catuaí Vermelho IAC 44, a susceptible C. arabica cultivar. Variation in aggressiveness components was observed between and within localities. Isolates with longer incubation periods also tended to have longer latent periods, although there was only a low correlation between these two aggressiveness components (r² = 0.34, P = 2.2 × 10^?16). Low‐sporulating isolates also had significantly longer incubation and latent periods. The H. vastatrix population from Minas Gerais and Espírito Santo is structured by the formation of groups of individuals with differential level of aggressiveness. Our results indicate that the variation in aggressiveness of the Brazilian H. vastatrix population may be associated with the geographic coffee‐producing areas.     

On the hunt for the alternate host of Hemileia vastatrix

Coffee leaf rust (CLR), caused by the fungal pathogen Hemileia vastatrix, has plagued coffee production worldwide for over 150 years. Hemileia vastatrix produces urediniospores, teliospores, and the sexual basidiospores. Infection of coffee by basidiospores of H. vastatrix has never been reported and thus far, no alternate host, capable of supporting an aecial stage in the disease cycle, has been found. Due to this, some argue that an alternate host of H. vastatrix does not exist. Yet, to date, the plant pathology community has been puzzled by the ability of H. vastatrix to overcome resistance in coffee cultivars despite the apparent lack of sexual reproduction and an aecidial stage. The purpose of this study was to introduce a new method to search for the alternate host(s) of H. vastatrix. To do this, we present the novel hypothetical alternate host ranking (HAHR) method and an automated text mining (ATM) procedure, utilizing comprehensive biogeographical botanical data from the designated sites of interests (Ethiopia, Kenya and Sri Lanka) and plant pathology insights. With the HAHR/ATM methods, we produced prioritized lists of potential alternate hosts plant of coffee leaf rust. This is a first attempt to seek out an alternate plant host of a pathogenic fungus in this manner. The HAHR method showed the highest‐ranking probable alternate host as Psychotria mahonii, Rubus apetalus, and Rhamnus prinoides. The cross‐referenced results by the two methods suggest that plant genera of interest are Croton, Euphorbia, and Rubus. The HAHR and ATM methods may also be applied to other plant–rust interactions that include an unknown alternate host or any other biological system, which rely on data mining of published data.     

Genetic Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

Suppression of Rust and Brown Eye Spot Diseases on Coffee by Phosphites and By‐products of Coffee and Citrus Industries

The rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively, are the most important fungal diseases on coffee in South America. Their management is mainly by chemical treatment, and there is no genetic resistance to brown eye spot known so far. Considering the need for developing alternative products for their control, the goal of this work was to evaluate the effects of phosphites and by‐products of coffee and citrus industries on rust and brown eye spot. Formulations of coffee and citrus industry by‐products, phosphites and their combination with fungicide were evaluated in field experiments, and their effect on fungal urediniospores and conidia was evaluated in vitro. In the field, treatments were applied individually or in combination and the in vitro assays were performed with manganese phosphite (Reforce Mn), potassium phosphite and citrus industry by‐product (Fortaleza), copper phosphite and coffee industry by‐product (Fitoforce Full), and fungicide. The severity and incidence of rust and brown eye spot on coffee leaves, yield, and leaf retention were evaluated in the field. Percentage of spore germination was evaluated in vitro for both fungi, whereas mycelial growth was evaluated for C. coffeicola only. The treatments Fortaleza, Reforce Mn and Fitoforce Full suppressed both diseases with a reduction in defoliation. In the year 2012, the plants treated with Reforce Mn and Reforce Mn + Fortaleza showed a yield increase of 72 and 88%, respectively, which was similar to the results shown by the fungicide treatment. In vitro inhibition of germination of H. vastatrix urediniospores and of C. coffeicola conidia was observed and suggests that the products exert some toxic effects to both fungi. Finally, the results observed indicate that the combined use of by‐products of plant‐processing industries and phosphites is an alternative and can be added efficiently to the management of coffee diseases.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

High-density genetic mapping for coffee leaf rust resistance

Coffee leaf rust caused by the fungus Hemileia vastatrix causes considerable economic losses for coffee producers. Although agrochemical products can provide sufficient disease control, the use of resistant cultivars is a safer alternative. This resistance may be constrained by one or a few genetic factors, mainly those found in material originating from interspecific hybrids. In this study, the genetic analysis of an F ₂ population consisting of 224 plants derived from a crossing of Híbrido de Timor UFV 427-15 (resistant) with Catuaí Amarelo IAC 30 (susceptible) showed that a dominant gene confers the resistance of coffee to race II of H. vastatrix. From a genetic map saturated with 25 amplified fragment length polymorphism (AFLP) markers linked to the resistance gene, we developed a high-density genetic map with six sequence-characterized amplified region (SCAR) markers delimiting a chromosomal region of 9.45 cM and flanking the dominant gene at 0.7 and 0.9 cM. This is the first saturated and high-density genetic map obtained from this region containing the resistance gene. The results of this study are of great importance for the introduction of molecular markers for marker-assisted selection; they will also facilitate studies related to the cloning, structure, and function of race-specific genes involved in the resistance of coffee trees to H. vastatrix.     

Tuber Blight Development in Potato Cultivars in Response to Different Genotypes of Phytophthora infestans

Migrations or introduction of new genotypes of Phytophthora infestans to a specific region imposes a different perspective for potato production. During 2009–2010, a late blight epidemic affected the Northeastern United States, which quickly spread through several states. The epidemic was characterized by the appearance of a new genotype of P. infestans designated US‐22, which was isolated from tomato and potato. Potato tubers are an essential component of late blight epidemics where the pathogen cannot overwinter on Solanaceous plants. Six potato cultivars were inoculated with 12 isolates of P. infestans (five different genotypes), including isolates of the genotype US‐22. Tuber blight development was characterized in terms of tissue darkening expressed as area under the disease progress curve values and lenticel infection. The responses indicated that US‐8 was more aggressive than US‐22, but US‐22 isolates obtained from potato were more aggressive on potato than those acquired from tomato. Tuber periderm responses to infection were limited, yet US‐8 isolates infected the periderm more often than US‐22 isolates. There were significant differences among the cultivars tested but cv. Jacqueline Lee was the most resistant overall. Although isolates of P. infestans genotype US‐22 were less aggressive in comparison with US‐8 isolates, US‐22 isolates still infected potato tubers and were as aggressive us US‐8 isolates on some cultivars. Management of late blight caused by isolates of US‐22 through host resistance may be feasible but imposes a different set of criteria for consideration from those that US‐8 imposed.     

Morphological and Pathogenic Characterization of Genetically Diverse Sclerotinia Isolates from European Red Clover Crops (Trifolium Pratense L.)

Clover rot, an important disease in European red clover crops, is caused by Sclerotinia trifoliorum or Sclerotinia sclerotiorum. Until today, little is known about the variation in aggressiveness among Sclerotinia isolates from red clover. Aggressiveness has never been correlated with morphological characteristics. Rapidly growing isolates may be more aggressive, but this was never investigated in S. trifoliorum before. Also nothing is known about the link between sclerotia production and aggressiveness. Oxalic acid is an important pathogenicity factor in Sclerotinia species, but its effect on aggressiveness is unknown in S. trifoliorum isolates. For this study, we selected 30 Sclerotinia isolates from 25 locations Europe: 26 S. trifoliorum isolates and 4 S. sclerotiorum isolates from two locations in France (Fr.A and Fr.B). For each isolate, the in vitro growth speed, sclerotia production, oxalate production and aggressiveness were analysed and correlations were estimated between aggressiveness and the other characteristics. Aggressiveness was assessed in vitro on detached leaves and in a greenhouse on young plants. Our isolates differed significantly in growth speed, sclerotia production, oxalate production and aggressiveness. The infections on detached leaves and young plants revealed interaction between isolates and plant genotypes and between isolates and cultivars, but there was no indication that pathotypes exist. In vitro growth speed and in vitro aggressiveness on detached leaves were positively correlated with aggressiveness on young plants, while sclerotia production was negatively correlated with aggressiveness on young plants. These factors can be used as predictors of aggressiveness of Sclerotinia isolates from red clover crops.     

Genetic variability of Fusarium wilt pathogen isolates of chickpea (Cicer arietinum L.) assessed by molecular markers

Genetic variability among 43 isolates of Fusarium oxysporum f.sp. ciceri, the chickpea wilt pathogen, collected from nine states of India including the four well-characterized races of the pathogen were assessed using the molecular markers, RAPDs and AFLP. Principal coordinate analysis of the similarity index data generated from the molecular marker studies mostly gave three different clusters: Of these two clusters represented race-1 and race-2, and the third cluster consisted of race-3 and race-4 pathogen isolates. In RAPDs a fourth cluster was seen which did not go with any of the four races of the pathogen. The molecular markers established the distinctness of race-1 and race-2 pathogen isolates and the close similarity of pathogen isolates of race-3 with that of race-4. AFLP was found to be more informative as it differentiated more number of the pathogen isolates with the known races with minimum of outliers. The high levels of DNA polymorphism observed with the molecular markers suggest the rapid evolution of new recombinants of the pathogen in the chickpea growing fields. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification and Characterization of the Causal Agent of Gummy Stem Blight from Muskmelon and Watermelon in East China

Gummy stem blight (GSB) is one of the most destructive foliar diseases of cucurbits, worldwide. To identify and characterize the pathogen which causes GSB on watermelon and muskmelon in East China, morphological characteristics, pathogenicity assays as well as sequence characterization of the rDNA internal transcribed spacer (ITS) were performed on 41 isolates collected from Jiangsu, Zhejiang, Anhui and Jiangxi provinces. The mycelia of all the isolates were white on top and olivaceous green to black with concentric circles at the bottom on potato‐dextrose agar medium. The isolates differed significantly on aggressiveness based on pathogenicity assays. rDNA‐ITS sequences and phylogenetic analysis confirmed the isolates as Didymella bryoniae. The isolates were found to be highly identical with the exception of 13 isolates, which had a guanine substitution instead of adenine at position 131 of the ITS.     

Characterization of Kenyan Isolates of Fusarium udum from Pigeonpea [Cajanus cajan (L.) Millsp.] by Cultural Characteristics, Aggressiveness and AFLP Analysis

Isolates of Fusarium udum from pigeonpea (Cajanus cajan) plants with wilt symptoms were collected from various districts in Kenya and were characterized using cultural characteristics, aggressiveness and amplified fragment length polymorphism (AFLP). The 56 isolates of F. udum showed a high level of variability in aerial mycelia growth, pigmentation and radial mycelia growth (colony diameter) on potato dextrose agar. The aggressiveness of 17 isolates of F. udum on seven pigeonpea varieties varied and five aggressive groups were observed in the present study. There were no relationships among cultural characteristics and aggressiveness. AFLP analysis of the 56 isolates was tested for genetic variability using seven primer combinations. A total of 326 fragments was generated of which 121 were polymorphic. Ten AFLP groups were identified among the Kenyan isolates and, although they were not genetically distinct, six AFLP subgroups were genetically distinct. AFLP had no relationship with cultural characteristics, aggressiveness and geographical origin of the isolates. This is the first report on the study of genetic variability of F. udum using DNA analysis.     

Variability of Colletotrichum kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

Twenty‐six isolates of Colletotrichum kahawae, the causal agent of coffee berry disease, from coffee in Africa, and 25 isolates, mostly of Colletotrichum gloeosporioides, from coffee and other tropical perennial crops, were examined for the ability to metabolize citrate and tartrate and their molecular genetic variability was assessed using restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR). Twenty‐four isolates of C. kahawae were also assessed using amplified fragment length polymorphisms (AFLP). Vegetative compatibility within a collection of nine isolates, including two of C. gloeosporioides was also assessed. All isolates of C. kahawae from across Africa failed to metabolize citrate or tartrate, but all other isolates metabolized one or both. Colletotrichum kahawae isolates also showed minimal variability using the molecular techniques with two isolates from Cameroon showing slightly different banding patterns in RFLP analysis. All other isolates had variable VNTR and RFLP banding patterns. AFLP analysis failed to detect variability within 12 isolates from Kenya, but did detect differences between isolates from other countries. Five isolates from Kenya were vegetatively compatible but differed from two from Cameroon and from two C. gloeosporioides isolates. Results demonstrate some geographic variability within C. kahawae isolates, although this is small, probably due to the relatively young age of C. kahawae populations. The biochemical and molecular techniques used showed clear differences from other Colletotrichum isolates, and can be used to distinguish the species. Lack of citrate and tartrate metabolism provides a readily applicable diagnostic method.     

Reaction of Some Coffea arabica Genotypes to Strains of Colletotrichum kahawae, the Cause of Coffee Berry Disease

Pathogenicity tests were performed on 11 genotypes of Coffea arabica using single‐isolate suspensions of Colletotrichum Kahawae obtained from 90 monoconidial isolates. The objective of this study was to estimate the proportion of pathogenic variation corresponding 10 differences in aggressiveness and virulence (races). A large part of the variation in the pathogen population was due to aggressiveness. The differential effects were too small to suggest conclusively that races exist. This paper discusses the possible causes for the observed small differential interaction and suggests breeding strategies that not only prevent possible adaptation of the pathogen to resistant varieties but also limit variation for resistance due to differences in aggressiveness of the pathogen.     

Cultural and Aggressiveness Variability of Cercospora coffeicola

Although brown eye spot of coffee, caused by Cerco‐spora coffeicola, is important for coffee production in Brazil, there is a general lack of knowledge regarding the disease. In this study, we evaluated the variability of both the cultural and aggressiveness traits of 60 isolates from coffee plants grown under conventional and organic systems in three regions of Minas Gerais State, Brazil. Variability among the isolates was detected with regard to all of the traits and was unrelated to an effect of either the region or cropping system. Mycelial growth, cercosporin production and sporulation were assessed in the laboratory. Of the 60 isolates, 27 did not sporulate at 25°C; the mycelial growth of all of the isolates and cercosporin production by 18 of the isolates linearly increased as the temperature rose from 18 to 26°C. We inoculated six selected isolates on plants of two coffee cultivars (‘Catuaí Vermelho IAC44’ and ‘Catucaí Vermelho 785‐15’) and evaluated the incubation period (IP), latent period (LP) and disease severity. All three of these traits were affected by temperature postinoculation and KCl amendment. The significant correlations were as follows: IP and LP in both cultivars; severity and leaf fall in both cultivars; and cercosporin production in vitro and severity values in ‘Catucaí Vermelho 785‐15’. In conclusion, we found that (i) C. coffeicola is highly variable for both cultural and aggressiveness traits; (ii) laboratory and glasshouse experiments were suitable to assess the pathogen variability; (iii) research protocols should account for the effect of environmental factors, such as temperature and KCl, on the traits evaluated; and (iv) these protocols should include the assessment of the IP instead of the LP, as both are correlated, and the IP is easier to evaluate.     

Pathogenicity and its alternation with both morphological and genetic variation in Plasmopara halstedii (sunflower downy mildew)

Morphological, pathogenic and genetic variation was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of several races using five singlezoosporangium isolates per pathogen isolate. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Genetic relationships were detected between the single zoosporangium isolates using 12 expressed sequence tags (EST)-derived markers. Analysis of the five single zoosporangium isolates for P. halstedii isolates showed variability within pathogen isolates for all aggressiveness criteria, but not for all pathogen isolates. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than the isolate of races 7xx. All pathogen isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria. There was no intra-genetic variation for all pathogen isolates, but it was observed an important genetic variation between single zoosporangium isolates of all races. No correlation was detected between pathogenicity traits and EST genotypes.     

Investigating variability and behaviour of Colletotrichum gloeosporioides strains from lesions of coffee blister spot

Coffee blister spot has been associated with species from the Colletotrichum genus, but there is no information on the variability of isolates present on leaf lesions. This study evaluated a population of Colletotrichum gloeosporioides strains from blister spot lesions in Coffea arabica. Colletotrichum spp. isolates were collected from blister spot lesions on leaves of coffee trees from Catuaí and Topázio cultivars (Coffea arabica). Monosporic cultures were obtained from colonies with sporulation. A pathogenicity test was carried out by inoculation of pathogens on the leaves of young coffee plants. C. gloeosporioides strains were characterized by morphologial, cytological and physiological analyses. The molecular analysis was carried out using Inter‐Retrotransposon Amplified Polymorphism (IRAP) markers. C. gloeosporioides strains showed no pathogenicity on coffee plants and presented a wide variability in all traits evaluated. The presence of sexual strains, formation of CATs (conidial anastomosis tubes) among conidial strains and high mycelial compatibility among strains observed suggest the occurrence of sexual and asexual recombination. The role of these C. gloeosporioides strains on the lesions of coffee plant leaves is unclear.     

Pathogenic and Genetic Variation among the Isolates of Rhizoctonia solani (AG 1‐IA), the Rice Sheath Blight Pathogen

Sheath blight disease of rice caused by Rhizoctonia solani is one of the most dreaded plant diseases faced by the rice farmers all over the world. None of the commercially cultivated rice varieties have sufficient level of field resistance, and the disease is presently being managed by chemical pesticides. In this study, 40 isolates of rice sheath blight pathogen, collected from diverse rice ecosystems from 12 different states of India, were characterized for their morphological, pathological and genetic variation. The isolates showed wide morphological variation in terms of size of sclerotia and abundance of sclerotia production. The virulence of each pathogen isolate was studied on four rice varieties, that is TN1, IR 64, Tetep and Swarnadhan in glasshouse, and observations were taken by measuring the relative lesion height. The relative lesion heights produced by these isolates on four different rice varieties varied widely. Genetic variation of the isolates was analysed using ISSR markers. The primers based on AG, GA, AC and CA repeats were informative and revealed polymorphism among the isolates. The polymorphism information content (PIC) of the primers ranged from 0.80 to 0.96, while the resolving power (Rp) ranged from 3.7 to 15.35. Largely, grouping of the isolates happened based on their geographical origin. One isolate from Titabar, Assam, and another from Adialabad, Telangana, were quite distinct from rest of the isolates.     

Resistance inducers applied alone or in association with fungicide for the management of leaf rust and brown eye spot of coffee under field conditions

The use of resistance inducers is a promising development in the management of plant diseases, owing to their ability to control a broad spectrum of pathogens and improve the efficacy of fungicides. This study evaluates different sources of phosphonates (potassium, manganese, copper), a formulation prepared from the by‐products of the coffee industry (Greenforce CuCa), as well as the effects of their application, alone or in association with fungicide, in the management of two important coffee fungal diseases in Brazil: leaf rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively. The effect of these products on defoliation, productivity and chemical composition of coffee beans (content of trigonelline, chlorogenic acid, caffeine and total soluble solids) was evaluated. Among all the alternative products tested individually, potassium phosphonate (P₂O₅—33.6% + K₂O—29%) stood out, particularly for rust control, which was similar to the results of fungicide treatments. Treatments with fungicide, Greenforce CuCa and cuprous oxide, individually, caused less plant defoliation. Regarding the chemical composition of the coffee beans, the manganese phosphonate treatment showed the highest values for trigonelline, chlorogenic acid, caffeine and total soluble solid content. The results of this study show that resistance inducers can be useful in disease management, may come to eventually replace traditional fungicides and can also contribute to the beverage quality.     

Study on Rosellinia necatrix Isolates Causing White Root Rot Disease in Taiwan

White root rot is a serious soil‐borne disease of several woods and crops. Recently, white root rot of tea shrubs and ornamental trees has increasingly been observed in Taiwan. Thirty‐six isolates of white root rot pathogen, showing pear‐shape swellings adjacent to the hyphal septa, had been isolated from samples of white root rot collected from Taiwan for about 4 years. The pathogen isolates produced Dematophora anamorph. Conidia of the pathogen were one‐celled, hyaline, subglobal, with truncate base, 2.9–5.8 × 1.9–3.5 μm . Ascospore dimensions were in the range of 37.0–55.0 × 5.4–7.9 μm with a short, longitudinal and straight germ slit, which complied with Rosellinia necatrix. Based on molecular studies, the pathogen isolates collected from Taiwan except R701 were identified as R. nectarix. Isolate R701, which was relatively polymorphic in internal transcribed spacer DNA sequence than other isolates, was temporarily considered as R. necatrix‐related pathogenic Rosellinia spp. All the tea cuttings (Camellia sinensis) inoculated with isolates developed typical white root rot symptoms. Pathogenicity tests demonstrated the presence of variation in virulence among the Rosellina isolates. Most of the R. necatrix isolates originating from Acer morrisonense were less virulent than those that originated from other hosts. The pathogenic Rosellinia spp., isolate R701, was also highly virulent to both cultivars of tea cuttings.