Virulence Diversity of Phakopsora pachyrhizi Isolates From East Africa Compared to a Geographically Diverse Collection.

Soybean rust, caused by the biotrophic pathogen Phakopsora pachyrhizi, is a highly destructive disease causing substantial yield losses in many soybean producing regions throughout the world. Knowledge about P. pachyrhizi virulence is needed to guide development and deployment of soybean germplasm with durable resistance against all pathogen populations. To assess the virulence diversity of P. pachyrhizi, 25 isolates from eight countries, including 17 isolates from Africa, were characterized on 11 soybean genotypes serving as differentials. All the isolates induced tan lesions with abundant sporulation on genotypes without any known resistance genes and on soybean genotypes with resistance genes Rpp4 and Rpp5b. The most durable gene was Rpp2, where 96% of the isolates induced reddish brown lesions with little or no sporulation. Of the African isolates tested, the South African isolate was the most virulent, whereas those from Kenya, Malawi, and some of the isolates from Tanzania had the lowest virulence. An Argentinian isolate was virulent on most host differentials, including two cultivars carrying multiple resistance genes. Ten distinct pathotypes were identified, four of which comprised the African isolates representing considerable P. pachyrhizi virulence. Soybean genotypes carrying Rpp1b, Rpp2, Rpp3, and Rpp5 resistance genes and cultivars Hyuuga and UG5 were observed to be resistant against most of the African isolates and therefore may be useful for soybean-breeding programs in Africa or elsewhere.

First Report of Phakopsora pachyrhizi Causing Rust on Soybean in Malawi.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has become established in Africa since the first report in Uganda in 1996 (2). The urediniospores, as windborne propagules, have infested new regions of Africa, initiating SBR in many countries, including Ghana and Democratic Republic of the Congo in 2007 (4) and Tanzania in 2014 (3). No refereed reports have been published about rust in Malawi, but some people have indicated that soybean rust may have been observed as early as 2008. Typical symptoms and signs of SBR, including leaf yellowing and tan, sporulating uredinia, were observed on soybean in May 2014 during field surveys in the major soybean-growing areas of Malawi, including the central (Dowa, Mchinji, and Kasungu) and southern (Thyolo) regions in nine out of 12 sites surveyed. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. Leaves exhibiting sporuliferous uredinia were sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The resulting DNA was analyzed by quantitative PCR using published Taqman assays for P. pachyrhizi and P. meibomiae, with a multiplexed exogenous internal control reaction to validate negative results (1). P. pachyrhizi DNA was detected in excess of 180,000 genome equivalents/cm2 in all samples, indicating a substantial infection. P. meibomiae DNA was determined to be absent from all samples, within the limit of quantification of ~2 pg DNA/cm2. Urediniospores dislodged from three leaves and inoculated onto susceptible soybean cultivar Williams 82 produced tan lesions after 2 weeks of incubation in a detached-leaf assay. This is the first confirmed report of P. pachyrhizi causing rust on soybean in Malawi, putting at risk 14,000 ha currently under soybean production. The reports of soybean rust in Malawi and adjoining countries will alter soybean production practices and research interests. In some cases, foliar application of fungicides has increased and planting dates have been changed to avoid conditions that are most conducive for rust development. Efforts to understand the virulence and genetic diversity of the pathogen in the region are needed in order to develop and deploy resistant cultivars. References: (1) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (2) R. Kawuki, et al. Afr. Crop Sci. J. 11:301, 2003. (3) H. M. Murithi et al. Plant Dis. 98:1586, 2014. (4) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

First Report of Phakopsora pachyrhizi on Soybean Causing Rust in Tanzania.

Phakopsora pachyrhizi Syd. was reported on legume hosts other than soybean in Tanzania as early as 1979 (1). Soybean rust (SBR), caused by P. pachyrhizi, was first reported on soybean in Africa in Uganda in 1996 (3), and its introduction into Africa was proposed to occur through urediniospores blowing from western India to the African east coastal areas by moist northeast monsoon winds (4). The fungus rapidly spread and was reported on soybean in South Africa in 2001, in western Cameroon in 2003, and in Ghana and the Democratic Republic of the Congo in 2007 (5). A second species causing SBR on soybean, P. meibomiae, has not been reported in Africa or elsewhere, outside of the Americas. From 2012 to 2014, symptomatic leaf samples were collected in the major soybean growing areas of the Tanzanian Southern Highlands (Iringa, Mbeya, and Ruvuma regions). Symptoms of SBR included yellowing of leaves and tan sporulating lesions. These symptoms were observed at flowering through seed maturity. From fields surveyed in 2012, 2013, and 2014, SBR was observed in 5 of 14, 7 of 11, and 14 of 31 fields, respectively. Some of the leaves sampled had up to 80% of the leaf area affected. When microscopically examined, urediniospores were elliptical, echinulate, and hyaline to pale yellowish brown. In 2014, sporuliferous uredinia were observed on leaf material collected from the Iringa and Ruvuma regions of Tanzania, and a subset of these samples was sent by APHIS permit to the University of Illinois. To confirm the pathogen, symptomatic soybean leaf tissue of approximately 1 cm2 was excised from each of the samples, and DNA was extracted using the FastDNA Spin Kit (MP Biomedicals, Solon, OH), with further purification using the MicroElute DNA Clean-up Kit (Omega Bio-Tek, Norcross, GA). The DNA was subjected to quantitative PCR using published Taqman assays for P. pachyrhizi, P. meibomiae, and a multiplexed exogenous internal control reaction to validate negative results (2). P. pachyrhizi DNA was detected in excess of 66,000 genome equivalents/cm2 in all samples, and P. meibomiae DNA was determined to be absent from all samples (limit of quantification ~2 pg DNA/cm2). Free surviving urediniospores were dislodged from 12 samples and inoculated onto susceptible soybean cultivar Williams 82, which produced sporulating SBR lesions after 2 weeks of incubation in a detached-leaf assay. Thus, Koch's postulates were completed. This is the first report of P. pachyrhizi causing rust on soybean in Tanzania. In vivo cultures have been established from most of these samples, and ongoing research includes an evaluation of the P. pachyrizi virulence on a differential set, and characterization of the genetic diversity. References: (1) D. L. Ebbels and D. J. Allen. Phytopath. Pap. 22:1-89. (2) J. S. Haudenshield and G. L. Hartman. Plant Dis. 95:343, 2011. (3) R. Kawuki et al. Afr. Crop Sci. J. 11:301, 2003. (4) C. Levy. Plant Dis. 89:669, 2005. (5) P. S. Ojiambo et al. Plant Dis. 91:1204, 2007.

First Report of Orange Rust Caused by Puccinia kuehnii in Sugarcane in Louisiana.

In June 2012, lesions typical of rust were observed on sugarcane cultivar Ho 05-961 (a complex hybrid of Saccharum L. spp.) on a farm near Schriever, Louisiana. Incidence and severity of disease symptoms were low. Two types of pustules were observed on leaves of the infected plants. One pustule type was reddish-brown in color turning brown with age, characteristic of brown rust which has been observed in Louisiana since 1979 (2). The other pustule type was orange and did not turn brown with age. Urediniospore samples from the two pustule types were collected. The morphology of the urediniospores from the reddish-brown pustules was consistent with that described for Puccinia melanocephala Syd. & Syd., the fungus that causes brown rust of sugarcane, while the morphology of the urediniospores from the orange pustules was consistent with those described for P. kuehnii E.J. Butler, the causal organism of orange rust of sugarcane (3). Telia and teliospores were not observed. The identity of the two species of Puccinia causing the brown and orange rust lesions was verified using the species-specific quantitative PCR assays (1). Two DNA samples extracted from the pustules identified as P. kuehnii were independently subjected to PCR amplification using primers Pk1F and Pk1R (1) to yield a product from the rDNA that was then bidirectionally sequenced using the same primers. The resulting 480-nt sequences were identical to each other, and a BLAST search of GenBank revealed 100% identity to 19 previously reported isolates of P. kuehnii but not more than 89% similarity to any isolate of P. melanocephala (4). To our knowledge, this is the first report of orange rust in Louisiana. In the 4 months following the detection of orange rust, observations of the disease have been limited to Ho 05-961. Seed cane increase plots of this newly released cultivar were surveyed, and orange rust symptoms and urediniospores were detected in 17 of 38 (45%) fields. The incidence and severity of the disease remained low, and the distribution appeared to be limited to the southern portion of the Louisiana sugarcane production area. References: (1) N. C. Glynn et al. Plant Pathol. 59:703, 2010. (2) H. Koike. Plant Dis. 64:226, 1980. (3) C. C. Ryan et al. Page 189 in: Diseases of Sugarcane: Major Diseases. C. Ricaud et al., eds. Elsevier, Amsterdam, 1989. (4) E. V. Virtudazo et al. Mycoscience 42:447, 2001.

A multiplexed immunofluorescence method identifies Phakopsora pachyrhizi Urediniospores and determines their viability.

Soybean rust, caused by Phakopsora pachyrhizi, occurs concomitantly wherever soybean is grown in the tropical and subtropical regions of the world. After reports of its first occurrence in Brazil in 2001 and the continental United States in 2004, research on the disease and its pathogen has greatly increased. One area of research has focused on capturing urediniospores, primarily by rain collection or wind traps, and detecting them either by microscopic observations or by immunological or molecular techniques. This system of detection has been touted for use as a potential warning system to recommend early applications of fungicides. One shortcoming of the method has been an inability to determine whether the spores are viable. Our study developed a method to detect viable P. pachyrhizi urediniospores using an immunofluorescence assay combined with propidium iodide (PI) staining. Antibodies reacted to P. pachyrhizi and other Phakopsora spp. but did not react with other common soybean pathogens or most other rust fungi tested, based on an indirect immunofluorescence assay using fluorescein isothiocyanate-labeled secondary antibodies. Two vital staining techniques were used to assess viability of P. pachyrhizi urediniospores: one combined carboxy fluorescein diacetate (CFDA) and PI, and the other utilized (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide] (FUN 1). Using the CFDA-PI method, viable spores stained green with CFDA and nonviable spores counterstained red with PI. Using the FUN 1 method, cylindrical intravacuolar structures were induced to form within metabolically active urediniospores, causing them to fluoresce bright red to reddish-orange, whereas dead spores, with no metabolic activity, had an extremely diffused, faint fluorescence. An immunofluorescence technique in combination with PI counterstaining was developed to specifically detect viable P. pachyrhizi urediniospores. The method is rapid and reliable, with a potential for application in forecasting soybean rust based on the detection of viable urediniospores.

First Report of Soybean Rust (Phakopsora pachyrhizi) on Florida Beggarweed (Desmodium tortuosum) in Alabama.

Soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi, was detected on Florida Beggarweed (Desmodium tortuosum) for the first time in Alabama in November, 2009. The pathogen was not observed in 2010 or 2011, probably because of the exceptionally dry, hot weather in the region. The pathogen was observed on multiple mature leaves of plants, evenly distributed through a field at the Wiregrass Research and Extension Center in Headland, Alabama, located in the southeast region of the state. Florida Beggarweed can serve as an overwintering host for SBR. Symptoms on leaves were consistent with SBR symptoms previously described on soybeans (1). Sori in multiple pustules were observed on the undersurface of the leaves. Urediniospores and paraphyses were observed microscopically and identified as P. pachyrhizi. Symptomatic leaves from 20 plants were analyzed using an Envirologix monoclonal antibody strip test kit at the Auburn University Plant Diagnostic Laboratory. A subsample of 20 plants were positive for the pathogen. Representative symptomatic leaves were sent to the USDA Molecular Diagnostic Laboratory in Beltsville, Maryland, for confirmation. DNA was extracted from sori aseptically removed from leaves using a Qiagen DNeasy Plant Mini Kit, and amplified with primers Ppa1 and NL4. The resulting partial ITS2 and 28S ribosomal RNA sequences were 100% identical to GenBank entry DQ354537. Voucher specimens were deposited in the USDA Agricultural Research Service, National Fungus Collection (BPI). To our knowledge, this is the first report of the disease on Florida Beggarweed in Alabama. References: (1) A. Carcamo Rodriguez et al. Plant Dis. 90:1260, 2006. (2) R. D. Frederick et al. Phytopathology 92:217, 2002.

First Report of Colletotrichum chlorophyti Causing Soybean Anthracnose.

Anthracnose of soybean [Glycine max (L.) Merr.] is caused by several Colletotrichum spp. (4). Petiole samples were collected from Alabama, Mississippi, and Illinois in 2009. Diseased tissues suspected of being caused by Colletotrichum were cut into 1- to 2-cm lengths, surface-disinfested, and placed on water agar. Pure cultures obtained by picking single spores from sporulating acervuli on tissue or hyphal tips on agar were transferred to acidic potato dextrose agar (APDA) at 24 ±1°C under 12-h cool-white fluorescent lighting. Isolates were grouped by morphological characteristics. One group consisting of six isolates (four from IL and one each from AL and MS) did not morphologically match any reported Colletotrichum spp. causing soybean anthracnose but matched the description of C. chlorophyti S. Chandra & Tandon (1,2). On APDA, colonies were initially pink, turning black after several days with smooth margins and no aerial mycelium. Conidial masses were light salmon in color. Conidia ranged from 15.5 to 21.3 µm long (mean 18.0 µm) × 2.5 to 4.3 wide (mean 3.3 µm) (n = 200). They were curved with tapered ends and a truncated base, aseptate, and hyaline. Chlamydospores were dark brown, clustered or chained together, and 5 to 12 µm wide (n = 30). Setae were straight, dark brown, and septate. Appressoria and perithecia were absent. Soybean plants (cv. Williams 82) at the V2 to V3 stage were atomized with a suspension of fragmented mycelia (40 mg/ml) using one isolate from IL. Plants were kept moist (>90% relative humidity) for 48 h in the dark, then transferred to normal growing conditions. Three days post-inoculation, younger trifoliolate leaf margins and intra- and interveinal lesions were necrotic surrounded by slight chlorosis. Isolations were obtained from symptomatic leaves and confirmed as C. chlorophyti by morphological characteristics. Further comparisons were completed with one isolate (IL1A or BPI 884117) by PCR and BLAST sequencing analyses of the partial ITS rDNA region, actin, ß-tubulin, GAPDH, and histone H3 genes (2) (GenBank Accession Nos. JX126475, JX126476, JX126477, JX126478, and JX126479, respectively). The results showed high identity of all the five sequences to two C. chlorophyti isolates, IMI 103806 and CBS 142.79 (Accession Nos. GU227894/GU227895 in ITS = 100%, GU227992/GU227993 in actin = 99%, GU228188/GU228189 in ß-tubulin = 99%, GU228286/GU228287 in GAPDH = 99% and 96%, respectively, and GU228090/GU228091 in histone H3 = 99%). Soybean anthracnose, commonly caused by C. truncatum, has curved and truncated conidia that are longer than C. chlorophyti. In addition, the two are distinguished by chlamydospores and lack of appressoria in C. chlorophyti combined with differences in multigene sequence analysis. Isolates of C. chlorophyti were reported to infect Chlorophytum sp. (Liliaceae) in India and Stylosanthes hamate in Australia (3). To our knowledge, there are no previous reports of this species in the United States or of it infecting soybean worldwide (3). This report describes C. chlorophyti as a novel incitant of soybean anthracnose. References: (1) S. Chandra and R. N. Tandon. Curr. Sci. 34:565, 1965. (2) U. Damm et al. Fungal Divers. 39:45, 2009. (3) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , May 21, 2012. (4) G. L. Hartman et al. Compendium of Soybean Diseases, APS Press, St. Paul, MN. pp. 13, 1999.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Pachyrhizus erosus in the United States.

Soybean rust, caused by the fungus Phakopsora pachyrhizi, was detected on jicama (Pachyrhizus erosus L. Urban) for the first time in the United States in November 2009. The pathogen was observed on leaves of a single, potted jicama plant grown outdoors in a residential area and on leaves of all plants in a 12-m2 demonstration plot located at the Auburn University Teaching Garden in Auburn, AL. Symptoms on the upper leaf surfaces were isolated chlorotic areas near the leaf edges in the lower part of the canopy. The abaxial surface was first observed to exhibit brown lesions and subsequently produced volcano-shaped uredinia. These symptoms are consistent with a rust previously described on jicama in Mexico (1). Representative symptomatic plant tissue was sent to the USDA National Identification Services (Mycology) Laboratory in Beltsville, MD for diagnostic confirmation at both the Urbana, IL lab and the USDA National Plant Germplasm and Biotechnology Laboratory for DNA testing. From an infected leaf, samples of approximately 5 mm2 were excised from a microscopically observed rust lesion and an apparently noninfected area. Total DNA was purified with the FastDNA Spin Kit (MP Biomedicals, Solon, OH) followed by the E.Z.N.A. MicroElute DNA Clean-Up Kit (Omega Bio-tek, Inc, Doraville, GA) per manufacturer's instructions. Detection of P. pachyrhizi and P. meibomiae DNA was achieved by quantitative PCR using the method of Frederick et al. (2) and a DNA standard of previously prepared P. pachyrhizi spores. The observed rust pustule was found to contain P. pachyrhizi DNA in excess of 28,000 genomes, while no P. pachyrhizi DNA was observed from the asymptomatic sample. Both samples were negative for P. meibomiae. The fungal structures present were confirmed to be Phakopsora spp. DNA was extracted from sori aseptically removed from leaves with a Qiagen (Valencia, CA) DNeasy Plant Mini Kit and amplified with primers Ppa1 and NL4. The resulting partial ITS2 and 28S ribosomal RNA sequences were 100% identical to GenBank entry DQ354537 P. pachyrhizi internal transcribed spacer 2 and 28S ribosomal RNA gene, partial sequence. Sequences from jicama from Alabama were deposited in GenBank. Voucher specimens were deposited in the USDA Agricultural Research Service, National Fungus Collection (BPI). To our knowledge, this is the first report of the disease on jicama in the United States. References: (1) A. Cárcamo Rodriguez et al. Plant Dis. 90:1260, 2006. (2) R. D. Frederick et al. Phytopathology 92:217, 2002.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Kudzu (Pueraria montana var. lobata) in Illinois.

Soybean rust, caused by Phakopsora pachyrhizi Syd., first was observed in the continental United States during 2004 on soybean (Glycine max (L.) Merr.) in Louisiana (4), and on kudzu (Pueraria montana (Lour.) Merr. var. lobata (Willd.) Maesen & Almeida) in Florida (2). Kudzu is a leguminous weed that is prevalent in the southern United States with its range extending northward into other states including Illinois. In October 2009, a kudzu patch located in Pulaski County in southern Illinois was investigated for the presence of soybean rust. Twenty-five leaflets were collected, and the abaxial sides of leaflets were evaluated visually for the presence of uredinia with a dissecting microscope. Uredinia and urediniospores were found on two leaflets. When viewed with a compound microscope, urediniospores were hyaline, echinulate, and measured 20 × 25 µm. On the basis of uredinia and urediniospores, the disease tentatively was identified as soybean rust caused by P. pachyrhizi. To confirm the identification, one leaflet with pustules was assayed with a Soybean Rust QuickStix Diagnostic Kit (Envirologix, Portland, ME). For the other leaflet, the area of the pustule was excised (approximately 28 mm2) and an area of the leaflet at the margin on the opposite half of the leaflet with no visible pustule (approximately 54 mm2) was excised. DNA was extracted from the excised areas of the leaflet for confirmation by quantitative PCR (Q-PCR) using primers and probe specific to P. pachyrhizi and P. meibomiae (Arthur) Arthur (1). Both the QuickStix Diagnostic Kit and the Q-PCR confirmed the diagnosis as soybean rust caused by P. pachyrhizi. Q-PCR also suggested the presence of a nonsporulating latent rust infection on the same kudzu leaflet at the margin on the opposite side of the midrib. Soybean rust first was confirmed on soybean in Illinois in 2006 (3), but to our knowledge, this is the first observation of the disease on kudzu in the state. This report confirms that at least some kudzu plants in Illinois are susceptible to soybean rust and that latent kudzu infection may exist without outward signs of the fungus. Currently, this is the most northern observation of soybean rust on kudzu in North America. It is unknown what role, if any, Illinois kudzu will play in the epidemiology of soybean rust in the state. Since kudzu tops die after the first frost, there is no expectation of P. pachyrhizi to overwinter in Illinois on kudzu as it does in some states adjacent to the Gulf of Mexico. References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) P. F. Harmon et al. Online publication. doi:10.1094/PHP-2005-0613-01-RS. Plant Health Progress, 2005. (3) G. L. Hartman et al. Plant Dis. 91:466, 2007. (4) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Diversity among isolates of squash mosaic virus.

cDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87% sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.

Correlation between arrested secondary plasmodesmal development and onset of accelerated leaf senescence in yeast acid invertase transgenic tobacco plants.

Mature leaves of a transgenic tobacco plant (Nicotiana tabacum L. var. Samsun, line A41-10) that constitutively express a yeast-derived acid invertase gene develop symptoms which are characterized by the presence of greenish-yellow and green sectors in the same leaf, and onset of early leaf senescence. Previous studies indicated that invertase activity was two- to threefold higher in the greenish-yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish-yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase-expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.