Effect of Selected Biopesticides in Reducing Soybean Rust (Phakopsora pachyrhizi) Development.

The intensive use of fungicides in controlling soybean rust (SBR), a damaging foliar fungal disease of soybean caused by the obligate fungus Phakopsora pachyrhizi, may have accelerated the insensitivity of P. pachyrhizi populations to fungicides. The objective of this study was to determine the effect of selected biopesticides and their application time on reducing SBR infection. There were differences (P < 0.05) in percent rust reduction values for application times, biopesticide treatments, and their interaction in detached-leaf and whole-plant greenhouse experiments. All application times and nearly all biopesticide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Among the treatments, Bacillus subtilis QST 713 and acibenzolar-S-methyl often reduced fungal sporulation more than the other treatments in detached-leaf and whole-plant greenhouse experiments. The identification of biopesticides effective to P. pachyrhizi may be a valuable alternative or complement to synthetic fungicides and may be useful in integrated pest management programs for SBR control.

Sensitivity of Phakopsora pachyrhizi Isolates to Fungicides and Reduction of Fungal Infection Based on Fungicide and Timing of Application.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is a damaging foliar fungal disease in many soybean-growing areas of the world. Strategies to manage SBR include the use of foliar fungicides. Fungicide types, the rate of product application, and the number and timing of applications are critical components for successful rust management. The objectives of this study were to determine i) the sensitivity of P. pachyrhizi isolates collected in the U.S. to a range of fungicides and ii) the reduction of fungal infection based on fungicide type and timing of applications on soybean. There were differences (P < 0.05) in effective concentration (EC50) values among the fungicides tested. Azoxystrobin had low EC50 values for both urediniospore germination and fungal sporulation on inoculated leaflets. There were differences (P < 0.05) in fungal sporulation for application times, fungicide treatments, and their interaction when the fungus was inoculated on plants. All application times and nearly all fungicide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Information on fungicide sensitivity of P. pachyrhizi isolates and the preventive and curative effects of different fungicides are important in the management of SBR.

Use of Quantitative Traits to Assess Aggressiveness of Phakopsora pachyrhizi Isolates from Nigeria and the United States.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important foliar diseases of soybean worldwide. The soybean-P. pachyrhizi interaction is often complex due to genetic variability in host and pathogen genotypes. In a compatible reaction, soybean genotypes produce tan-colored lesions, whereas in an incompatible reaction soybean genotypes produce an immune response (complete resistance) or reddish-brown lesions (incomplete resistance). In this study, in total, 116 and 72 isolates of P. pachyrhizi from Nigeria and the United States, respectively, were compared based on six quantitative traits to assess their aggressiveness on two soybean genotypes. All isolates produced reddish-brown lesions on plant introduction (PI) 462312 and tan lesions on TGx 1485-1D. The number of days after inoculation to first appearance of lesions, uredinia, and sporulation, along with the number of lesions and sporulating uredinia per square centimeter of leaf tissue, and the number of uredinia per lesion, were significantly (P < 0.001) different between the two soybean genotypes for all isolates from each country. The number of days to first appearance of lesions, uredinia, and sporulation were greater on PI 462312 than on TGx 1485-1D for all the test isolates. Similarly, the number of lesions and sporulating uredinia per square centimeter, and the number of uredinia per lesion were lower on PI 462312 than on TGx 1485-1D. For both soybean genotypes, the number of sporulating uredinia per square centimeter significantly (P = 0.0001) increased with an increase in the number of lesions per square centimeter. Although the slope of the regression of sporulating uredinia on number of lesions was greater (P < 0.0001) when TGx 1485-1D was inoculated with Nigerian isolates compared with U.S. isolates, slopes of the regression lines did not differ significantly (P > 0.0675) when PI 46312 was inoculated with Nigerian or U.S. isolates. This is the first study that used a large number of isolates from two continents to assess aggressiveness of P. pachyrhizi using multiple traits in soybean genotypes with contrasting types of disease reaction.

Identification of Species of Botryosphaeriaceae Causing Bot Gummosis in Citrus in California.

Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as 'Eureka' lemon, 'Valencia', 'Washington Navel', 'Fukumoto', grapefruit, 'Satsuma', and 'Meyer' lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, ß-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.

Identification and Pathogenicity of Fungal Pathogens Associated with Stem-End Rot of Avocado in California.

Stem-end rot of harvested avocado fruit commonly occurs wherever the crop is cultivated. Multiple fungal species have been described as causal agents. To determine the causal pathogens of stem-end rot in California, fungal isolations were conducted from symptomatic fruit, and fungi were identified by morphological and molecular techniques. In 2010 and 2011, a total of 177 isolates were recovered from 290 avocado fruit collected from seven orchards in one of the major avocado growing areas in Southern California. The majority of isolates was identified as Neofusicoccum luteum (65%), with the remainder either as Colletotrichum gloeosporioides (33%) or Phomopsis sp. (2%). In a pathogenicity test, N. luteum caused significantly (P < 0.05) more severe stem-end rot than either C. gloeosporioides or Phomopsis sp. No significant (P > 0.05) differences in stem-end rot severity were observed between inoculations with N. luteum isolated from fruit stem-end rot and N. luteum or N. parvum isolated from branch cankers. This confirms that stem-end rot of avocado can be initiated by fungi causing branch cankers. Although low humidity and rainfall during much of the growing and harvest seasons in California are considered unfavorable conditions for the development of avocado stem-end rot, the identification of the causal pathogens is of value when decays have to be managed during outbreaks, and it stresses the importance of managing branch cankers.

Effect of Fungicide Application on the Management of Avocado Branch Canker (Formerly Dothiorella Canker) in California.

Members of the Botryosphaeriaceae family have been associated with branch cankers of avocado trees (Persea americana) in California. Canker infections are initiated by spores entering the host plant through fresh wounds such as pruning wounds. With high-density planting becoming more common in the California avocado industry, more intensive pruning may increase the occurrence of branch canker. The objective of this study was to evaluate the preventive ability of some commercial fungicides belonging to different chemical families against fungal pathogens associated with avocado branch canker. Initially, 12 fungicides were tested in vitro for their effect on the inhibition of mycelial growth of three isolates of Dothiorella iberica and isolates (five per species) of Neofusicoccum australe, N. luteum, N. parvum, and Phomopsis sp. Subsequently, azoxystrobin, fludioxonil, metconazole, and pyraclostrobin, selected because of their low effective concentrations that reduce 50% of mycelial growth (EC50 values), and myclobutanil, selected for its high EC50 value, were tested in two field experiments. Azoxystrobin and fludioxonil were used in a premix with propiconazole and cyprodinil, respectively, in field trials. Significant differences (P < 0.05) were observed among fungicides in field trials. Azoxystrobin + propiconazole had the highest percent inhibition at 52 and 62% (internal lesion length) in trial 1 and trial 2, respectively, although this level of inhibition was not significantly different from that of metconazole. A significant correlation (r = 0.51, P < 0.05) was observed between internal lesion length data in the field experiment and EC50 data from in vitro fungicide screening. Application of azoxystrobin + propiconazole and metconazole can play a key role in protecting Californian avocado against fungi causing avocado branch canker.

A Cut-Stem Inoculation Technique to Evaluate Soybean for Resistance to Macrophomina phaseolina.

Charcoal rot of soybean is caused by the fungal pathogen Macrophomina phaseolina. Effective and reliable techniques to evaluate soybean for resistance to this fungus are needed to work toward a management scheme that would utilize host resistance. Three experiments were conducted to investigate the use of a cut-stem inoculation technique to evaluate soybean genotypes for resistance to M. phaseolina. The first experiment compared aggressiveness of M. phaseolina isolates collected from soybean on different soybean genotypes. Significant (P < 0.05) differences among the isolates and genotypes for relative area under disease progress curve (RAUDPC) were found without a significant isolate-genotype interaction. The second experiment compared 14 soybean genotypes inoculated with M. phaseolina in multiple trials conducted in two environments, the greenhouse and growth chamber. Significant (P < 0.05) differences among environments and highly significant (P < 0.001) differences among soybean genotypes for RAUDPC were found. The environment-genotype interaction was nonsignificant (P > 0.05). Soybean genotypes DT97-4290, DT98-7553, DT98-17554, and DT99-16864 had significantly (P < 0.05) lower RAUDPC than 7 of the 14 genotypes. The third experiment evaluated resistance in selected Phaseolus spp. and soybean genotypes. The range of RAUDPC for Phaseolus spp. was similar to that of soybean. The Phaseolus lunatus 'Bush Baby Lima' had significantly (P < 0.05) lower RAUDPC than P. vulgaris genotypes evaluated. The cut-stem inoculation technique, which has several advantages over field tests, successfully distinguished differences in aggressiveness among M. phaseolina isolates and relative differences among soybean genotypes for resistance to M. phaseolina comparable with results of field tests.

First Report of a Fusarium sp. and Its Vector Tea Shot Hole Borer (Euwallacea fornicatus) Causing Fusarium Dieback on Avocado in California.

Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 µm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 µm, 9.2 to 17.2 × 3.4 to 4.8 µm, and 13.5 to 17.6 × 4.3 to 4.7 µm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Koch's postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California. References: (1) W. Danthanarayana. Tea Quarterly 39:61, 1968. (2) H. Kajimura and N. Hijii. Ecol. Res. 7:107, 1992; (3) Mendel et al., Phytoparasitica, DOI 10.1007/s12600-012-0223-7, 2012. (4) R. J. Rabaglia. Annals Entomol. Soc. Amer. 99:1034, 2006.

Pathogenic Variation of Phakopsora pachyrhizi Isolates on Soybean in the United States from 2006 to 2009.

The introduction of Phakopsora pachyrhizi, the cause of soybean rust, into the United States is a classic case of a pathogen introduction that became established in a new geographical region overwintering on a perennial host (kudzu, Pueraria lobata). The objective of our study was to classify the pathogenic variation of P. pachyrhizi isolates collected in the United States, and to determine the spatial and temporal associations. In total, 72 isolates of P. pachyrhizi collected from infected kudzu and soybean leaves in the United States were purified, then established and increased on detached soybean leaves. These isolates were tested for virulence and aggressiveness on a differential set of soybean genotypes that included six genotypes with known resistance genes (Rpp), one resistant genotype without any known characterized resistance gene, and a susceptible genotype. Three pathotypes were identified among the 72 U.S. P. pachyrhizi isolates based on the virulence of these isolates on the genotypes in the differential set. Six aggressiveness groups were established based on sporulating-uredinia production recorded for each isolate on each soybean genotype. All three pathotypes and all six aggressiveness groups were found in isolates collected from the southern region and from both hosts (kudzu or soybean) in 2008. Shannon's index based on the number of pathotypes indicated that isolates from the South region were more diverse (H = 0.83) compared with the isolates collected in other regions. This study establishes a baseline of pathogenic variation of P. pachyrhizi in the United States that can be further compared with variation reported in other regions of the world and in future studies that monitor P. pachyrhizi virulence in association to deployment of rust resistance genes.

Dynamics of Soybean Rust Epidemics in Sequential Plantings of Soybean Cultivars in Nigeria.

Soybean rust, caused by Phakopsora pachyrhizi, is an important foliar disease of soybean. Disease severity is dependent on several environmental factors, although the precise nature of most of these factors under field conditions is not known. To help understand the environmental factors that affect disease development, soybean rust epidemics were studied in Nigeria by sequentially planting an early-maturing, highly susceptible cultivar, TGx 1485-1D, and a late-maturing, moderately susceptible cultivar, TGx 1448-2E, at 30- to 45-day intervals from August 2004 to September 2006. Within each planting date, disease onset occurred earlier on TGx 1485-1D than on TGx 1448-2E, and rust onset was at least 20 days earlier on soybean planted between August and October than on soybean planted between November and April. The logistic model provided a better description of the temporal increase in rust severity than the Gompertz model. Based on the logistic model, the highest absolute rates of disease increase were observed on soybean planted in September 2006 and October 2004 for TGx 1485-1D and TGx 1448-2E, respectively. Disease severity as measured by the relative area under disease progress curve (RAUDPC) was significantly (P < 0.05) negatively correlated with evaporation (r = -0.73), solar radiation (r = -0.59), and temperature (r = -0.64) but positively correlated with urediniospore concentration (r = 0.58). Planting date and soybean cultivar significantly (P < 0.05) affected disease severity, with severity being higher on soybean crops planted during the wet season than those planted in the dry season. Within the wet season, planting in May and July resulted in a significantly (P < 0.05) lower RAUDPC than planting between August and October. Yields were significantly (P < 0.001) related to RAUDPC during the wet season, whereby an increase in RAUDPC resulted in a linear decrease in yield. This study suggests that selection of planting date could be a useful cultural practice for reducing soybean rust.

Culturing Phakopsora pachyrhizi on Detached Leaves and Urediniospore Survival at Different Temperatures and Relative Humidities.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important foliar diseases of soybean worldwide. In a series of experiments, multiple objectives were addressed to determine the (i) longevity of detached soybean leaves, (ii) reproductive capacity of uredinia on leaves inoculated and/or incubated on the abaxial versus adaxial side of the leaf, (iii) reproductive capacity of uredinia and urediniospore germination when spores were harvested at regular intervals or all at once, and (iv) effect of temperature and relative humidity (RH) on urediniospore germination. A detached-leaf assay using agar medium amended with 6-benzylaminopurine performed better in retarding leaf chlorosis than filter paper alone among five soybean genotypes. Among the three susceptible genotypes tested, detached leaves of cv. Williams 82 had the lowest level of leaf chlorosis and often allowed for the greatest urediniospore production and germination rate. Temperature and RH played significant roles in survival of urediniospore as measured by germination rates. Viable urediniospores were harvested from infected soybean leaves maintained at room temperature (23 to 24°C at 55 to 60% RH) for up to 18 days, whereas freshly harvested urediniospores that were desiccated for 12 h before being placed in vials and maintained at room temperature remained viable for up to 30 days. Urediniospore hydration was the major factor for the dormancy reversion; thermal shock with hydration and no thermal shock with hydration treatments had consistently similar urediniospore germination rates. In the RH experiment, urediniospores harvested from inoculated leaf pieces maintained at 85% RH had the highest germination rates compared with higher and lower RH. Improvement in P. pachyrhizi cultural techniques and understanding of urediniospore survival will enhance our knowledge of the pathogen biology, host-plant relationship, and conditions that favor the infection, reproduction, and survival of the pathogen.

Pathogenic variation of Phakopsora pachyrhizi infecting soybean in Nigeria.

Soybean rust, caused by Phakopsora pachyrhizi, is an important disease in Nigeria and many other soybean-producing countries worldwide. To determine the geographical distribution of soybean rust in Nigeria, soybean fields were surveyed in the Derived Savanna (DS), Northern Guinea Savanna (NGS), and Southern Guinea Savanna (SGS) agroecological zones in Nigeria between 2004 and 2006. Disease severity in each zone was determined and analyzed using geostatistics. Prevalence of infected fields and disease severity in surveyed fields were significantly (P < 0.05) different between geographical zones with both variables being higher in the DS zone than in either NGS or SGS zones. Geostatistical analysis indicated that the spatial influence of disease severity at one location on severity at other locations was between 75 and 120 km. An exponential model best described the relationship between semivariance and lag distance when rust severity was high. Spatial interpolation of rust severity showed that locations in the DS zone were more conducive for the rust epidemic compared to areas in the NGS zone. In the 2005 survey, 116 purified isolates were established in culture on detached soybean leaves. To establish the nature of pathogenic variation in P. pachyrhizi, a set of four soybean accessions with Rpp(1), Rpp(2), Rpp(3), and Rpp(4) resistance genes, two highly resistant and two highly susceptible genotypes were inoculated with single uredinial isolates. Principal component analysis on the number of uredinia per square centimeter of leaf tissue for 116 isolates indicated that an adequate summary of pathogenic variation was obtained using only four genotypes. Of these four, PI 459025B (with Rpp(4) gene) and TGx 1485-1D had the lowest and highest number of uredinia per square centimeter, respectively. Based on cluster analysis of the number of uredinia per square centimeter, seven pathotype clusters were determined. Isolates in cluster III were the most virulent, while those in cluster IV were the least virulent. Shannon's index (H) revealed a more diverse pathogen population in the DS zone (H = 1.21) compared to the rust population in SGS and NGS with H values of 1.08 and 0.91, respectively. This work will be useful in breeding and management of soybean rust by facilitating identification of resistant genotypes and targeting cultivars with specific resistance to match prevailing P. pachyrhizi pathotypes in a given geographical zone.

Evaluation of Soybean Germplasm for Resistance to Soybean Rust (Phakopsora pachyrhizi) in Nigeria.

Soybean rust, caused by Phakopsora pachyrhizi, is one of the most important constraints to soybean production worldwide. The absence of high levels of host resistance to the pathogen has necessitated the continued search and identification of sources of resistance. In one set of experiments, 178 soybean breeding lines from the International Institute of Tropical Agriculture were rated for rust severity in the field in 2002 and 2003 at Ile-Ife, Yandev, and Ibadan, Nigeria. Thirty-six lines with disease severity ≤3 (based on a 0-to-5 scale) were selected for a second round of evaluation in 2004 at Ibadan. In the third round of evaluation under inoculated field conditions, 11 breeding lines with disease severity ≤2 were further evaluated for rust resistance at Ibadan in 2005 and 2006. The breeding lines TGx 1835-10E, TGx 1895-50F, and TGx 1903-3F consistently had the lowest level of disease severity across years and locations. In another set of experiments, 101 accessions from the United States Department of Agriculture-Agricultural Research Service and National Agriculture Research Organization (Uganda) were evaluated in the first round in 2005 under inoculated conditions in the screenhouse; 12 accessions with disease severity ≤20% leaf area infected were selected for evaluation in the second round in 2005 and 2006 under inoculated field conditions at Ibadan. Highly significant differences (P < 0.0001) in disease severity were observed among the 101 accessions during this first round of rust evaluation. Significant (P < 0.0001) differences in rust severity and sporulation also were observed among the 12 selected accessions. Accessions PI 594538A, PI 417089A, and UG-5 had significantly (P < 0.05) lower disease severity than all other selected accessions in both years of evaluation, with rust severities ranging from 0.1 to 2.4%. These results indicate that some of the breeding lines (TGx 1835-10E, TGx 1895-50F, and TGx 1903-3F) and accessions (PI 594538A, PI 417089A, and UG-5) would be useful sources of soybean rust resistance genes for incorporation into high-yielding and adapted cultivars.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi in Ghana.

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 µm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 µl of spore suspension (1 × 106 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 µg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 µm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

Rapid Screening of Musa Species for Resistance to Black Leaf Streak Using In Vitro Plantlets in Tubes and Detached Leaves.

This study investigated the utility of inoculation of in vitro plantlets in tubes and detached leaves as reliable and rapid assays for screening Musa genotypes against Mycosphaerella fijiensis, the causal agent of black leaf streak. In the first part of the study, three types of inocula were evaluated to determine suitability for in vitro inoculation. Inoculation of in vitro plantlets with mycelial fragments resulted in significantly (P < 0.05) higher levels of disease severity and faster rates of disease progress compared with inoculations using conidial suspensions. In the detached leaf assay, amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color. Leaf pieces on medium containing gibberellic acid at 5 mg/liter had about 5% chlorosis at 52 days after plating. When in vitro plantlets in tubes and detached leaves of 10 Musa genotypes with different levels of disease resistance were inoculated with M. fijiensis, there were significant (P < 0.05) differences among genotypes in leaf area infected, incubation time, and symptom evolution time. For incubation time and leaf area infected, cultivars responded depending on their level of disease resistance, with resistant genotypes Calcutta-4 and PITA-17 having significantly (P = 0.001) longer incubation times and lower infected leaf areas compared with the susceptible cultivar Agbagba and moderately resistant cultivar FHIA-23. A similar pattern in cultivar response was observed for symptom evolution time. Leaf area infected was not significantly (P = 0.2817 for two-tailed t test) different when assessed using the two assays, and infected leaf areas in both assays were strongly correlated (r = 0.88, n = 48, P < 0.0001). Although incubation times were significantly (P = 0.0062 for two-tailed t test) different between the two assays, values from the two assays were strongly correlated (r = 0.69, n = 48, P < 0.0001). These results show that these two assays are rapid and space-effective, and can reliably be used for screening Musa genotypes for resistance to black leaf streak.

Comparison of Field, Greenhouse, and Detached-Leaf Evaluations of Soybean Germplasm for Resistance to Phakopsora pachyrhizi.

Fourteen soybean accessions and breeding lines were evaluated for resistance to soybean rust caused by the fungus Phakopsora pachyrhizi. Evaluations were conducted in replicated experiments in growth chambers using detached leaves and under greenhouse and field conditions. In growth-chamber experiments, inoculation of detached leaves with 1 × 106 spores/ml resulted in a significantly (P < 0.0001) higher total number of pustules and spores per unit leaf area than inoculations with lower spore concentrations. Amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color in detached leaves. Leaf pieces on a medium containing kinetin at 10 mg/liter had 5% chlorosis at 18 days after plating compared with leaf pieces on media amended with all other plant hormones, which had higher levels of chlorosis. Leaf age significantly affected number of pustules (P = 0.0146) and number of spores per pustule (P = 0.0088), and 3- to 4-week-old leaves had a higher number of pustules and number of spores per pustule compared with leaves that were either 1 to 2 or 5 to 6 weeks old. In detached-leaf and greenhouse screening, plants were evaluated for days to lesion appearance, days to pustule formation, days to pustule eruption, lesion number, lesion diameter, lesion type, number of pustules, and spores per pustule in 1-cm2 leaf area. Plants also were evaluated for diseased leaf area (in greenhouse and field screening) and sporulation (in field screening) at growth stage R6. There were significant (P < 0.0001) differences among genotypes in their response to P. pachyrhizi infection in the detached-leaf, greenhouse, and field evaluations. Accessions PI 594538A, PI 417089A, and UG-5 had very low levels of disease compared with the susceptible checks and all other genotypes. Detached-leaf, greenhouse, and field results were comparable, and there were significant correlations between detached-leaf and greenhouse (absolute r = 0.79; P < 0.0001) and between detached-leaf and field resistance (absolute r = 0.83; P < 0.0001) across genotypes. The overall results show the utility of detached-leaf assay for screening soybean for rust resistance.

First Report of Rust Caused by Phakopsora pachyrhizi on Soybean in Democratic Republic of Congo.

Nigeria (1) and Uganda (3) are the closest countries to the Democratic Republic of Congo (DRC) where soybean rust caused by Phakopsora pachyrhizi has been reported. In February 2007, during a disease survey in DRC, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 10 fields in the following areas in Bas Congo Province: Bangu, Kimpese, Kolo-Fuma, Lukala, Mbanza-Ngungu, Mpalukide, Mvuazi, and Ntemo. Rust incidence in these fields ranged from 85 to 100%, while severity ranged between 3 and 35% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 16 to 20 µm. Within a week of collection, infected leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) for pathogen identification. DNA was extracted from sections of leaves containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 10 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Infected leaves of cultivar Vuangi collected from one field each in the INERA Research Station, Kimpese-Crawford, and Kimpese-Ceco were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves of susceptible cultivar TGx 1485-1D (4). Lesions on inoculated leaves developed 5 days after inoculation (DAI), and pustules (110 to 130 µm) formed 7 DAI and erupted 2 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculation of another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. Seedlings of cultivar Williams also showed typical rust symptoms when inoculated separately with urediniospores collected from nine fields (i.e., all except Kimpese-Ceco, which was infective in the detached leaf assay). Inoculation and incubation were carried out at the FDWSRU Plant Pathogen Containment Facility at Fort Detrick as described earlier (2). The PCR assay, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in DRC. To our knowledge, this is the first report of P. pachyrhizi infecting soybean in DRC. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) E. Kawuki et al. J. Phytopathol. 151:7, 2003. (4) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.