Jean C. Batzer

Expansion of the sooty blotch and flyspeck complex on apples based on analysis of ribosomal DNA gene sequences and morphology

Sooty blotch and flyspeck (SBFS) is a late-season disease of apple and pear fruit that cosmetically damages the cuticle, resulting in produce that is unacceptable to consumers. Previous studies reported that four species of fungi comprise the SBFS complex. We examined fungal morphology and the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA of 422 fungal isolates within the SBFS complex from nine orchards in four Midwestern states (USA) and compared them to previously identified species. We used LSU sequences to phylogenetically place the isolates at the order or genus level and then used ITS sequences to identify lineages that could be species. We used mycelial and conidial morphology on apple and in culture to delimit putative species. Thirty putative species found among the Midwest samples were shown to cause SBFS lesions on apple fruit in inoculation field trials. Among them Peltaster fructicola and Zygophiala jamaicensis have been associated previously with SBFS in North Carolina. The LSU analyses inferred that all 30 SBFS fungi from Midwestern orchards were Dothideomycetes; one putative species was within the Pleosporales, 27 were within Dothideales, and two putative species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella.

Diversity and Biogeography of Sooty Blotch and Flyspeck Fungi on Apple in the Eastern and Midwestern United States

Sooty blotch and flyspeck (SBFS) fungi on apple fruit were sampled from nine orchards in four midwestern U.S. states during 2000 and 30 orchards in 10 eastern U.S. states during 2005 in order to estimate taxonomic diversity and discern patterns of geographic distribution. Forty apple fruit per orchard were arbitrarily sampled and colonies of each mycelial phenotype were counted on each apple. Representative colonies were isolated, cultures were purified, and DNA was extracted. For representative isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions of ribosomal DNA were amplified and sequenced. In total, 60 SBFS putative species were identified based on ITS sequences and morphological characteristics; 30 of these were discovered in the 2005 survey. Modified Kochs postulates were fulfilled for all 60 species in an Iowa orchard; colonies resulting from inoculation of apple fruit were matched to the original isolates on the basis of mycelial type and ITS sequence. Parsimony analysis for LSU sequences from both surveys revealed that 58 putative SBFS species were members of the Dothideomycetes, 52 were members of the Capnodiales, and 36 were members of the Mycosphaerellaceae. The number of SBFS species per orchard varied from 2 to 15. Number of SBFS species and values of the Margalef and Shannon indexes were significantly (P < 0.05) lower in 21 orchards that had received conventional fungicide sprays during the fruit maturation period than in 14 unsprayed orchards. Several SBFS species, including Schizothyrium pomi, Peltaster fructicola, and Pseudocercosporella sp. RH1, were nearly ubiquitous, whereas other species, such as Stomiopeltis sp. RS5.2, Phialophora sessilis, and Geastrumia polystigmatis, were found only within restricted geographic regions. The results document that the SBFS complex is far more taxonomically diverse than previously recognized and provide strong evidence that SBFS species differ in geographic distribution. To achieve more efficient management of SBFS, it may be necessary to understand the environmental biology of key SBFS species in each geographic region.

A New View of Sooty Blotch and Flyspeck

Sooty blotch and flyspeck (SBFS) fungi colonize the surface wax layer of the fruit of apple, pear, persimmon, banana, orange, papaya, and several other cultivated tree and vine crops. In addition to colonizing cultivated fruit crops, SBFS fungi also grow on the surfaces of stems, twigs, leaves, and fruit of a wide range of wild plants. The disease occurs worldwide in regions with moist growing seasons. SBFS is regarded as a serious disease by fruit growers and plant pathologists because it can cause substantial economic damage. The smudges and stipples of SBFS often result in downgrading of fruit from premium fresh-market grade to processing use. This review describes the major shifts that have occurred during the past decade in understanding the genetic diversity of the SBFS complex, clarifying its biogeography and environmental biology, and developing improved management strategies.

Obtaining weather data for input to crop disease-warning systems: leaf wetness duration as a case study

Os sistemas de alerta fitossanitario sao ferramentas de suporte a decisao desenvolvidos para ajudar os agricultures a determinar o melhor momento da aplicacao das medidas de controle para combater as doencas de plantas. As variaveis meteorologicas sao dados de entrada quase que obrigatorios desses sistemas. Este trabalho apresenta uma revisao sobre os meios pelos quais as variaveis meteorologicas sao coletadas para serem usadas como dados de entrada em sistemas de alerta fitossanitario e sobre os desafios associados a logistica de obtencao desses dados. Essa revisao compara o monitoramento meteorologico ao nivel do produtor, nas propriedades agricolas, com aquele feito ao nivel de redes de estacoes meteorologicas, assim como discute as vantagens e desvantagens entre medir e estimar tais variaveis meteorologicas. Especial enfase e dada a duracao do periodo de molhamento foliar (DPM), nao somente pela sua importância como dado de entrada em diversos sistemas de alerta fitossanitario, mas tambem pelo desafio de se obter dados acurados dessa variavel. Pode-se concluir, apos ampla discussao do assunto, que nao ha um metodo unico e melhor para se obter os dados meteorologicos para uso em sistemas de alerta fitossanitario; por outro lado, as circunstâncias a nivel local, regional e nacional provavelmente influenciam a estrategia de maior sucesso.

Novel fungal genera and species associated with the sooty blotch and flyspeck complex on apple in China and the USA.

Fungi in the sooty blotch and flyspeck (SBFS) complex cause blemishes on apple and pear fruit that result in economic losses for growers. The SBFS fungi colonise the epicuticular wax layer of pomaceous fruit but do not invade the cuticle. Fungi causing fuliginous and punctate mycelial types on apple are particularly difficult to identify based on morphological criteria because many species in the SBFS complex share the same mycelial phenotypes. We compared the morphology and nuclear ribosomal DNA phylogeny (ITS, LSU) of 11 fungal strains isolated from SBFS blemishes on apple obtained from two provinces in China and five states in the USA. Parsimony analysis, supported by cultural characteristics and morphology in vitro, provided support to delimit the isolates into three novel genera, representing five new species. Phaeothecoidiella, with two species, P. missouriensis and P. illinoisensis, is introduced as a new genus with pigmented endoconidia in the Dothideomycetes. Houjia (Capnodiales) is introduced for H. pomigena and H. yanglingensis. Although morphologically similar to Stanjehughesia (Chaetosphaeriaceae), Houjia is distinct in having solitary conidiogenous cells. Sporidesmajora (Capnodiales), based on S. pennsylvaniensis, is distinguished from Sporidesmium (Sordariomycetes) in having long, multiseptate conidiophores that frequently have a subconical, darkly pigmented apical cell, and very long, multi-euseptate conidia.

Dissoconiaceae associated with sooty blotch and flyspeck on fruits in China and the United States

Zasmidium angulare, a novel species of Mycosphaerellaceae, and several novel taxa that reside in Dissoconiaceae, were identified from a collection of apples and Cucurbita maxima (cv. Blue Hubbard) from China and the USA that exhibited sooty blotch and flyspeck (SBFS) signs on their host substrata. Morphology on fruit surfaces and in culture, and phylogenetic analyses of the nuclear ribosomal DNAs 28S and internal transcribed spacer regions, as well as partial translation elongation factor 1-alpha gene sequences in some cases, were used to delineate seven previously unidentified species and three known species. Pseudoveronaea was established as a new genus of Dissoconiaceae, represented by two species, P. ellipsoidea and P. obclavata. Although Pseudoveronaea was morphologically similar to Veronaea, these fungi clustered with Dissoconiaceae (Capnodiales) rather than Chaetothyriales (Herpotrichiellaceae). Ramichloridium mali comb. nov., and three novel species, R. cucurbitae, R. luteum and R. punctatum were closely related with R. apiculatum, which together formed a distinct subclade in Dissoconiaceae. Species of Dissoconium s.lat. clustered in two well-supported clades supported by distinct morphological and cultural features. Subsequently Uwebraunia, a former synonym of Dissoconium, was resurrected for the one clade, with new combinations proposed for U. australiensis, U. commune, U. dekkeri and U. musae. Furthermore, we also reported that D. aciculare, Dissoconium sp., U. commune and U. dekkeri were associated with SBFS on apples.

Phylogenetic placement of plant pathogenic Sclerotium species among teleomorph genera.

Phylogenetic analyses and morphological characteristics were used to assess the taxonomic placement of eight plant-pathogenic Sclerotium species. Members of this genus produce only sclerotia and no fruiting bodies or spores, so Sclerotium species have been difficult to place taxonomically. Sequences of rDNA large subunit (LSU) and internal transcribed spacer (ITS) regions were determined for isolates of Sclerotium cepivorum, S. coffeicola, S. denigrans, S. hydrophilum, Ceratorhiza oryzae-sativae, S. perniciosum, S. rhizodes, S. rolfsii and S. rolfsii var. delphinii. Parsimony analysis grouped two species previously thought to be in the Basidiomycota, S. denigrans and S. perniciosum, within the Ascomycota; these species were found to have affinities with the teleomorph genera Sclerotinia and Stromatinia and the asexual Sclerotium cepivorum, which was known earlier to be related to Sclerotinia species. The other Sclerotium species were placed in one of two basidiomycetous groups, genera Athelia or Ceratobasidium. Based on rDNA analysis and morphology the basidiomycetous Sclerotium hydrophilum and S. rhizodes were transferred to genus Ceratorhiza, the anamorph of Ceratobasidium species. Sclerotium coffeicola was found to be close to S. rolfsii var. delphinii and S. rolfsii var. rolfsii, which was shown earlier to have an Athelia teleomorph.

Diversity of sooty blotch and flyspeck fungi from apples in northeastern Turkey

Sooty blotch and flyspeck (SBFS) is a mid- to late-season fungal disease complex that blemishes apples and other tree fruit crops in humid regions worldwide. SBFS is a concern for apple growers in northeastern Turkey, but the composition of the SBFS species assemblage in Turkey is unknown. In this study, SBFS fungi were isolated from apples collected in 2008 in the Rize Province of northeastern Turkey. Pressed SBFS colonies with subtending apple cuticle were shipped to Iowa State University for isolation. Of 633 primary isolates from 148 apple peels, 87 cultures were purified, 67 isolates were genetically characterized and 33 isolates examined for morphology. The internal transcriber spacer (ITS) ribosomal DNA and a portion of the 28S large subunit region (LSU) were compared to previously identified fungi using parsimony analysis. Putative species were delineated from ITS sequences as well as morphology on apple and in culture. Twelve species were delineated based on parsimony analysis of ITS sequences and morphology. A newly discovered and described species from the survey was Scleroramularia abundans; newly discovered putative species included Zygophiala sp. FS3.3, Stomiopeltis spp. RS7.1 and RS7.2, and Chaetothyriales sp. F1; previously recovered putative species included Zygophiala sp. FS6 and Stomiopeltis sp. RS4.1; and previously discovered and named SBFS species included Schizothyrium pomi, Zygophiala wisconsinensis, Microcyclosporella mali, Microcyclospora tardicrescens, and Peltaster fructicola. Based on parsimony analysis of the LSU, one species was placed in the subclass Chaetothyriomycetidae and 11 species were placed in subclass Dothideomycetidae. Ten species were placed in the order Capnodiales. These findings expand the documented extent of genetic diversity within the worldwide SBFS complex and are the first published description of the composition of the SBFS complex from Turkey.

Spatial Heterogeneity of Leaf Wetness Duration in Apple Trees and Its Influence on Performance of a Warning System for Sooty Blotch and Flyspeck

To determine the effect of sensor placement on the performance of a disease-warning system for sooty blotch and flyspeck (SBFS), we measured leaf wetness duration (LWD) at 12 canopy positions in apple trees, then simulated operation of the disease-warning system using LWD measurements from different parts of the canopy. LWD sensors were placed in four trees within one Iowa orchard during two growing seasons, and in one tree in each of four orchards during a single growing season. The LWD measurements revealed substantial heterogeneity among sensor locations. In all data sets, the upper, eastern portion of the canopy had the longest mean daily LWD, and was the first site to form dew and the last to dry. The lower, western portion of the canopy averaged about 3 h less LWD per day than the top of the canopy, and was the last zone where dew formed and the first to dry off. On about 25% of nights when dew occurred in the top of the canopy, no dew formed in the lower, western canopy. Intracanopy variability of LWD was more pronounced when dew was the sole source of wetness than on days when rainfall occurred. Daily LWD in the upper, eastern portion of the canopy was slightly less than reference measurements made at a 0.7-m height over turfgrass located near the orchard. When LWD measurements from several canopy positions were input to the SBFS warning system, timing of occurrence of a fungicide-spray threshold varied by as much as 30 days among canopy positions. Under Iowa conditions, placement of an LWD sensor at an unobstructed site over turfgrass was a fairly accurate surrogate for the wettest part of the canopy. Therefore, such an extra-canopy LWD sensor might be substituted for a within-canopy sensor to enhance operational reliability of the SBFS warning system.

Improving sooty blotch and flyspeck severity estimation on apple fruit with the aid of standard area diagrams

Sooty blotch and flyspeck is caused by numerous species of fungi that colonize the surface of apple fruit and thereby lower its market value. Although this disease poses a substantial threat to apple growers’ profitability in some regions, reliable and cost-effective methods for epidemiological and disease control studies have not been validated, nor are they widely available. We modified a standard area diagram to aid sooty blotch and flyspeck severity assessments and quantified its impact on accuracy and precision of visual estimates. Samples of ‘Fuji’ and ‘Mutsu’ fruit were photographed both from the top and laterally. Severity was assessed from a sub-sample of 160 images using image analysis software. Validation of the diagram was performed by eight raters who independently assessed severity in two series of selected images representing the lateral view and the top view, initially unaided and subsequently with the aid of the scale. Severity estimates ranged from 0.4% to 98% (most fruit had <10% severity). Accuracy and precision of the estimates were significantly improved when using the diagrammatic scale; concordance correlation coefficient values increased from 0.81 to 0.95. A strong tendency to underestimate severity for the mid-range to high levels was minimized when using the aid, which also improved reproducibility of the estimates among raters. In addition to strengthening evidence that a standard area diagram can be used reliably in sooty blotch and flyspeck studies, we expanded its application to disease assessment in the peduncle region, which enhances the usefulness of the method for evaluating efficacy of management practices.