Alternative Hosts in the Families Poaceae and Cyperaceae for Xanthomonas vasicola pv. vasculorum, Causal Agent of Bacterial Leaf Streak of Corn.

The bacterial pathogen Xanthomonas vasicola pv. vasculorum was first reported in the United States causing bacterial leaf streak on Nebraska corn (Zea mays) in 2016. The bacterium is also known to cause disease in sugarcane, grain sorghum, broom bamboo, and various palm species. The objective of this study was to identify alternative hosts for X. vasicola pv. vasculorum among plants commonly found in corn growing areas of the United States. In repeated greenhouse experiments, 53 species of plants found in the United States that had not been tested previously for susceptibility to X. vasicola pv. vasculorum were inoculated with the pathogen and monitored for symptom development. Eleven species in the family Poaceae exhibited symptoms: oat (Avena sativa), rice (Oryza sativa), orchardgrass (Dactylis glomerata), indiangrass (Sorghastrum nutans), big bluestem (Andropogon gerardii), little bluestem (Schizachyrium scoparium), timothy (Phleum pratense), sand bluestem (Andropogon hallii), green foxtail (Setaria viridis), bristly foxtail (Setaria verticillata), and johnsongrass (Sorghum halepense). Yellow nutsedge (Cyperus esculentus) in the Cyperaceae also was a symptomatic host. In addition, endophytic colonization by X. vasicola pv. vasculorum was found in three asymptomatic alternative hosts: downy brome (Bromus tectorum), tall fescue (Festuca arundinacea), and western wheatgrass (Pascopyum smithii). Experiments were also conducted in the field to determine the potential for alternative hosts to become infected by natural inoculum. Symptoms developed only in big bluestem and bristly foxtail in field experiments. These results suggest that infection of alternative hosts by X. vasicola pv. vasculorum can occur, but infection rates might be limited by environmental conditions.

Controlled release of complexed DNA from polycaprolactone film: comparison of lipoplex and polyplex release.

This report investigates the comparative in vitro controlled release and transfection efficiencies of pDNA-lipofectamine complex (lipoplex) and pDNA-poly(ethylene imine) complex (polyplex), from a biodegradable polycaprolactone (PCL) film. The effect of molecular weight of gelatin used as a porogen on in vitro release and transfection efficiency was also studied. A sustained release profile was obtained for naked pDNA and lipoplex from polymeric films for a month, while the release of polyplexes (PEI/DNA) is simply a burst at day 5, with little or no release thereafter. The release of polyplexes from PCL films is retarded due to interaction between the polyplexes and the polymer. A high burst release was seen for naked pDNA which was suppressed in the presence of gelatin. The extent of suppression of the burst effect by gelatin increased with its molecular weight. For complexed pDNA (lipoplex), the release was slow, but could be accelerated using gelatin; again the acceleration in release is dependant on the molecular weight of the gelatin used. The addition of gelatin as a porogen has no effect on the release of polyplexes from PCL films. The bioactivity of released plasmid DNA and complexes was studied by in vitro transfection using COS-7 cells. Transfection was observed from released lipoplexes samples till day 9 from PCL film with lower MW gelatin and till day 18 in the case of PCL films with higher MW gelatin. The results also showed that the bioactivity of released lipoplexes was superior to that of the naked pDNA.

An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control.

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

First Report of Leaf Spot of Smooth Bromegrass Caused by Pithomyces chartarum in Nebraska.

Smooth bromegrass (Bromus inermis Leyss.) is the most common perennial grass species cultivated for forage in North America. During late fall of 2004, smooth bromegrass plants in Lincoln, NE were observed to have brown lesions on leaf midveins that were several centimeters long. Symptomatic leaves were surface disinfested for 1 min in 2% NaOCl and incubated at 25°C on potato dextrose agar (PDA) and water agar. The fungus, Pithomyces chartarum (Berk. & Curt) Ellis, was isolated consistently and identified on the basis of morphological characteristics (1). Colonies were effused and black on PDA. Conidiophores measured 3.5 to 8 × 1.9 to 3.9 µm and were smooth and single. Conidia (7 to 25 × 9.5 to 14 µm) were broadly ellipsoidal, pale brown to dark brown, verrucose with mainly three transverse septa and one to two longitudinal septa. Pathogenicity tests were conducted on 50-day-old plants by spraying with a conidial suspension (2.5 × 105 spores per ml). Control plants were sprayed with sterile water. All plants were kept in a moist chamber (100% relative humidity) for 3 days and then transferred to a greenhouse (25°C, >70% relative humidity, and a 12-h photoperiod). One week after spraying, elongated lesions developed on leaf midveins of inoculated plants from which P. chartarum was consistently reisolated. No symptoms were observed on control plants. While P. chartarum has been described as a saprotroph or a parasite on a wide range of plants primarily in the tropics and subtropics, including the southern United States (2), it was reported previously on B. inermis only in Canada (3). This report expands the distribution and host range of P. chartarum as a pathogen in the United States. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (2) D. F. Farr et al. Fungal Databases, Systematic Botany and Mycology Laboratory, On-line publication. ARS, USDA, 2005. (3) J. H. Ginns. Compendium of Plant Disease and Decay Fungi in Canada 1960-1980. Res. Br. Can. Agric. Publ. 1813, 1986.

First Report of Sudden Death Syndrome of Soybean Caused by Fusarium solani f. sp. glycines in Nebraska.

During August of 2004, soybean (Glycine max (L.) Merr.) plants exhibiting symptoms typical of sudden death syndrome (SDS) caused by Fusarium solani (Mart.) Sacc. f. sp. glycines (= Fusarium virguliforme Akoi, O'Donnell, Homma, & Lattanzi) (1) were observed in Nemaha and Pierce counties in eastern Nebraska. Leaf symptoms ranged from small chlorotic spots to prominent interveinal necrosis on plants at R5-R6 growth stages. Taproots of symptomatic plants were plated on potato dextrose agar (PDA) amended with hymexazol, ampicillin, and rifampicin (HAR). Resulting fungal isolates grew slowly and developed masses of blue macroconidia, characteristic of F. solani f. sp. glycines. Sorghum seed infested with the isolates were placed 1.5 cm below soybean seeds of the susceptible cv. Sloan planted in clay pots (3). Noninfested sorghum seed and sorghum seed infested with F. oxysporum were controls. Plants were maintained for 32 days at 27.5 ± 2.5°C in the greenhouse. Small cholorotic spots were observed on leaves of F. solani f. sp. glycines-inoculated plants within 21 days followed by the development of interveinal chlorosis. Roots of symptomatic plants were plated on PDA with HAR and F. solani f. sp. glycines was recovered. Identification of the fungal cultures was further confirmed as F. solani f. sp. glycines by a real-time quantitative polymerase chain reaction (qPCR) assay described by Gao et al. (2). During 2005, SDS symptoms were also reported in early planted soybeans from Jefferson and Seward counties and the presence of SDS was confirmed by qPCR. The confirmation of SDS at multiple locations suggests that the pathogen is widely distributed in the eastern one-third of Nebraska. SDS could be a serious threat to soybean production in this area since spring weather conditions favor SDS infection and many producers plant soybean early in cool soils. References: (1) T. Akoi et al. Mycologia 95:660, 2003. (2) X. Gao et al. Plant Dis. 88:1372, 2004. (3) K. W. Roy et al. Plant Dis. 81:259, 1997.

Influence of Lysobacter enzymogenes Strain C3 on Nematodes.

Chitinolytic microflora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells. Here, the influence of Lysobacter enzymogenes strain C3 on Caenorhabditis elegans, Heterodera schachtii, Meloidogyne javanica, Pratylenchus penetrans, and Aphelenchoides fragariae is described. Exposure of C. elegans to L. enzymogenes strain C3 on agar resulted in almost complete elimination of egg production and death of 94% of hatched juveniles after 2 d. Hatch of H. schachtii eggs was about 50% on a lawn of L. enzymogenes strain C3 on agar as compared to 80% on a lawn of E. coli. Juveniles that hatched on a lawn of L. enzymogenes strain C3 on agar died due to disintegration of the cuticle and body contents. Meloidogyne javanica juveniles died after 4 d exposure to a 7-d-old chitin broth culture of L. enzymogenes strain C3. Immersion of A. fragariae, M. javanica, and P. penetrans juveniles and adults in a nutrient broth culture of L. enzymogenes strain C3 led to rapid death and disintegration of the nematodes. Upon exposure to L. enzymogenes strain C3 cultures in nutrient broth, H. schachtii juveniles were rapidly immobilized and then lysed after three days. The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.

First Report of Crown and Stem Rot of Orchid (Orchis palustris) Caused by Sclerotinia minor.

Orchis palustris Jacq. is a wild orchid native to wetlands in eastern Anatolia. During June of 2003, near Erzurum, Turkey, a decline of this orchid was observed in several meadows that had been irrigated for forage production. Stems were chlorotic, wilted, and collapsed. There was a soft, watery rot at the crowns and lower stems. White mycelium and black sclerotia formed on necrotic stem and crown tissues. The fungus was isolated from sclerotia on potato dextrose agar (PDA) and identified as Sclerotinia minor Jagger on the basis of small sclerotia (0.5 to 2.5 mm long) scattered throughout the colonies (2). Pathogenicity was confirmed by inoculating stems of 8-week-old plants with mycelial plugs from 5-day-old PDA cultures and enclosing inoculated plants in transparent plastic bags for 3 days. After 2 weeks, symptoms similar to those in the field were observed, and S. minor was reisolated from inoculated plants. Noninoculated control plants remained asymptomatic. The disease was previously observed on O. laxiflora Lam. in Turkey (1), but to our knowledge, this is the first report of S. minor infecting O. palustris References: (1) C. Eken et al. Plant Pathol. 52:802, 2003. (2) L.M. Kohn. Phytopathology 69:881, 1979.

Hypericin and hypocrellin induced apoptosis in human mucosal carcinoma cells.

Potent photosensitizers hypocrellin A (HA), hypocrellin B (HB) and hypericin (HY) are lipid-soluble perylquinone derivatives of the genus Hypericum and have a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by HA, HB and HY are still unclear. Moreover, no reports have mentioned cell apoptosis induced by HA, HB and HY in human nasopharyngeal carcinoma (NPC) and other mucosal cells. In this study, we attempt to clarify the photodynamic effects of HA, HB and HY compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon and bladder cells. Using these cell lines we investigated few hallmarks of apoptotic commitments in a drug dose dependent manner. Tumor cells photo-activated with HA, HB and HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly(ADP-ribose) polymerase, a caspases substrate, showed the classical cleavage pattern (116 to 85 kDa) associated with apoptosis in HA, HB and HY-treated cell lysates. In addition, 85 kDa cleaved product was blocked by the tetrapepdide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. Both inhibitors protect tumor cells from apoptosis. These results demonstrate that tumor cell death induced by HA, HB and HY is mediated by caspase proteases. This study also identifies HB as a more potent and promising photosensitizer for the treatment of mucosal cancer cells.

Induction of apoptosis by Hypericin through activation of caspase-3 in human carcinoma cells.

Potent photosensitizer Hypericin (HY), is a lipid soluble perylquinone derivative of the genus Hypericum and has a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by this compound is still unclear. Furthermore, there are no reports on mechanisms in cell apoptosis induced by perylquinones in human nasopharyngeal carcinoma (NPC) and other mucosal cells. We studied the photodynamic effects of HY compound in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon (CCL-220.1) and bladder (SD) cells. Using these cell lines we investigated few hall marks of apoptotic commitments in a drug and light dose dependent manner. Tumor cells photoactivated with HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced in all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116-85 kDa) associated with apoptosis in PDT-treated cell lysates. In addition, 85 kDa cleaved product was blocked by using tetrapeptide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. These results demonstrate that tumor cell death induced by photoactivated HY is mediated by caspase proteases. This study also identifies that CNE2, CCL-220.1 (colon) and SD (bladder) cell lines are more sensitive than TW0-1 cell line to PDT using perylquinone HY.

Photodynamic-induced apoptosis of human nasopharyngeal carcinoma cells using Hypocrellins.

It has been reported that novel photosensitizers Hypocrellin A and B, lipid soluble perylquinone derivatives of the genus Hypericum have a strong photodynamic effect on tumors and viruses. The molecular mechanisms of tumor cell death induction by Hypocrellin A and B are poorly understood. In this study, we have examined the photodynamic effects of Hypocrellin A and B compounds in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells. Using these cell lines we investigated the role of the apoptotic pathway in photosensitized Hypocrellin A and B-mediated cell death. Tumor cells photoactivated with Hypocrellin A and B showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by both tumor cell lines as evidenced by the externalization of phosphatidylserine (PS). A dose-dependent increase in caspases-3 protease activity inhibitable by the tetrapeptide inhibitor DEVD-CHO was also observed in both cell lines. Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116 to 85 kDa) associated with apoptosis in Hypocrellin A and B-treated cell lysates. In addition, caspase inhibition blocked the externalization of membrane PS, indicating that the loss of membrane phospholipid asymmetry is a downstream event of caspases activation. These results demonstrate that tumor cell death induced by Hypocrellin A and B is mediated by caspase proteases. In conclusion, this study identifies both Hypocrellins (A and B) as potent and promising photosensitizers for the treatment of NPC.

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3.

ABSTRACT Stenotrophomonas maltophilia strain C3, a biocontrol agent of Bipolaris sorokiniana in turfgrass, produced chitinases in broth media containing chitin. Chitinases were partially purified from culture fluid by ammonium sulfate precipitation and chitin affinity chromatography. The chromatography fraction with the highest specific chitinase activity was inhibitory to conidial germination and germ-tube elongation of B. sorokiniana, but it was less inhibitory than the protein fraction or the raw culture filtrate. The fraction exhibited strong exochitinase and weak endo-chitinase activity. Optimum temperature and pH for chitinase activity were 45 to 50 degrees C and 4.5 to 5.0, respectively. Chitinase activity was inhibited by Hg(2+) and Fe(3+), but not by other metal ions or enzyme inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the chromatography fraction revealed the presence of five protein bands of 25, 32, 48, 65, and 75 kDa. Partial amino acid sequences of the 32-, 65-, and 75-kDa proteins indicated that they are homologous to known bacterial chitinases. There was no homology found in the partial amino acid sequences of the 25- and 48-kDa proteins to any known chitinases. Five chitinase-active proteins were detected in the protein and chromatography fractions by activity gels, but when each protein was extracted and re-electrophoresed separately under denaturing conditions, only 32- or 48-kDa proteins were revealed. It was concluded that strain C3 produces at least two chitinases that are antifungal.

The Role of Chitinase Production by Stenotrophomonas maltophilia Strain C3 in Biological Control of Bipolaris sorokiniana.

ABSTRACT The role of chitinase production by Stenotrophomonas maltophilia strain C3 in biological control of leaf spot on tall fescue (Festuca arundinacea), caused by Bipolaris sorokiniana, was investigated in vitro and in vivo. The filtrate of a broth culture of C3, with chitin as the carbon source, was separated into fractions. A high molecular-weight fraction (>8 kDa) was chitinolytic and more inhibitory than a low-molecular-weight, nonchitinolytic fraction to conidial germination and hyphal growth by B. sorokiniana and to leaf spot development. A protein fraction derived by ammonium sulfate precipitation and a chitinase fraction purified by chitin affinity chromatography also were chitinolytic and highly antifungal. The chitinolytic fractions caused swelling and vacuolation of conidia and discoloration, malformation, and degradation of germ tubes. When boiled, the chitinolytic fractions lost chitinase activity along with most of the antifungal properties. Two chitinase-deficient and two chitinase-reduced mutants of C3 were compared with the wild-type strain for inhibition of germination of B. sorokiniana conidia on tall fescue leaves and for suppression of leaf spot development in vivo. The mutants exhibited reduced antifungal activity and biocontrol efficacy, but did not lose all biocontrol activity. An aqueous extract of leaves colonized by wild-type C3 had higher chitinase activity than that of noncolonized leaves and was inhibitory to conidial germination. The addition of chitin to leaves along with the wild-type strain increased both chitinase and antifungal activity. The chitinase activity level of extracts from leaves colonized by a chitinase-deficient mutant of C3, with and without added chitin, was no higher than the background, and the extracts lacked antifungal activity. Chitinolysis appears to be one mechanism of biological control by strain C3, and it functions in concert with other mechanisms.

Biological Control of Bipolaris sorokiniana on Tall Fescue by Stenotrophomonas maltophilia Strain C3.

ABSTRACT Stenotrophomonas maltophilia strain C3 was evaluated for control of leaf spot on tall fescue (Festuca arundinacea) caused by Bipolaris sorokiniana. In growth chamber experiments, C3 inhibited conidial germination on leaf surfaces and reduced lesion frequency and percent diseased leaf area compared with nontreated controls. The amount of leaf spot suppression was related to the C3 dose applied. The highest dose tested, 10(9) CFU/ml, prevented nearly all B. sorokiniana conidia from germinating on treated leaf surfaces and provided nearly complete suppression of lesion development. When colloidal chitin was added to C3 cell suspensions of 10(7) or 10(8) CFU/ml, biocontrol efficacy was significantly increased over C3 applied alone, whereas addition of chitin to a C3 cell suspension of 10(9) CFU/ml had no effect. In field experiments, application of C3 to tall fescue turf resulted in significant reductions in infection frequency and disease severity compared with nontreated controls. Strain C3 applied at 10(9) CFU/ml was more effective than C3 applied at 10(7) CFU/ml, and amendment of the lower dose with colloidal chitin enhanced its efficacy. Populations sizes of C3 established on foliage in a growth chamber and in the field were directly related to dose applied. Chitin amendments did not affect C3 population size.

Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare.

Jasmonic acid has properties of a plant hormone, including the induction of specific genes associated with plant defense. We previously described jar1-1, an Arabidopsis jasmonate response mutant that exhibits reduced sensitivity to methyl jasmonate. We have further characterized this mutant and two new alleles; jar1-2 from a gamma irradiated population, and jar1-4 from a T-DNA mutant population. Seedling root growth in jar1-1 was equally insensitive to methyl jasmonate and jasmonic acid, indicating that the defect was not in the conversion of methyl jasmonate to the acid. None of the jar1 mutants showed an altered sensitivity to auxin, cytokinin, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicating that the lesion does not affect the general uptake or transport of hormones. A soil fungus, Pythium irregulare, was found to blight jar1-1. Cultures of this organism caused the symptoms in all three jar1 mutants but not in wild type, indicating that increased susceptibility was due to the lesion in the JAR1 locus. A fatty acid desaturase triple mutant that is defective in the biosynthesis of jasmonic acid (J. Browse, Washington State University) was also susceptible, confirming that jasmonate is involved in resistance. The jar1-1 locus was mapped to the lower end of chromosome 2, about 11.4 cM from as1 and 1.6 cM from cer8. These results establish that jasmonate signaling plays an important role in resistance to soil micro-organisms in plants.

A Sensitive ELISA for Pythium ultimum Using Polyclonal and Species-Specific Monoclonal Antibodies.

A double-antibody sandwich indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of Pythium ultimum. A polyclonal antibody produced to cell walls of P. ultimum was used as the capture antibody, while a P. ultimum-specific mono-clonal antibody (MAb E5) was used for recognition of the fungus. In the ELISA, culture extracts of 7 isolates of P. ultimum exhibited strong positive reactions, whereas none of the 37 isolates of other Pythium spp. and fungal genera had positive reactions. P. ultimum was detected by ELISA in roots of bean, cabbage, and sugar beet seedlings grown in pathogen-infested soil. ELISA optical density readings for infected bean and sugar beet root samples were highly correlated (r > 0.9) with infection levels determined by culturing the samples on water agar. The correlation between the two methods of testing cabbage roots was low, but all cabbage roots in which P. ultimum was detected by culturing were strongly positive in the ELISA. Samples of roots infected with P. irregulare and those with no Pythium infection did not react in the ELISA. The ELISA was highly sensitive; the fungus was detected in culture extracts diluted 1:5,000,000 and in roots with less than 1 infection per 100 cm root.

First Report of Pythium ultimum, P. irregulare, Rhizoctonia solani AG4, and Fusarium proliferatum from Arrowleaf Clover (Trifolium vesiculosum): A Disease Complex.

Poor stand establishment, failure to recover after grazing, and premature plant death have reduced the utilization of arrowleaf clover (Trifolium vesiculosum Savi) as a forage crop in the southeastern United States in recent years. Clover plants collected from poor stands in east Texas pastures during the 1995 to 1996 and 1996 to 1997 seasons first exhibited root disease symptoms as young seedlings in the fall. Symptoms consisted of one or more of the following: tan discoloration of lateral roots and taproot; root pruning; and small, tan, sunken lesions on the taproot and crown. Many Rhizobium nodules were brown and dead. Toward spring, symptoms increased in severity. Root lesions became larger and darker, and internal crown discoloration was observed. Disease incidence reached 100% in both growing seasons. Premature death of plants also was observed, especially in pastures where plants had been grazed. Most of the fungi isolated from diseased roots were Pythium, multi- and binucleate Rhizoctonia, and Fusarium spp. Many plants were infected with two or three pathogens simultaneously. Two Rhizoctonia isolates (AR96-17 and -26) were identified as R. solani anastomosis group 4 (AG4; D. R. Sumner, University of Georgia). AR96-8 was identified as Fusarium proliferatum (T. Matsushima) Nirenberg (Fusarium Research Center, Penn State University; deposited as isolate M-8382). Three Pythium isolates (AR96-7, -11 and -39) were identified as P. irregulare Buisman, based on oogonial and sporangial characteristics. Eight other Pythium isolates were not identified. Pythium isolate AR97-1, found in 1997, was identified as P. ultimum Trow. Greenhouse studies to confirm pathogenicity of these isolates were conducted by sowing cv. Yuchi arrowleaf clover seed into artificially infested soilless medium. In pathogenicity tests for P. ultimum, seedling emergence for controls was 69% after 8 days, but no seedlings emerged from P. ultimum-infested media. Several seeds showed imbibition and emerging radicles but were symptomatic. The pathogen was reisolated from necrotic radicles. After 19 weeks, survival and root disease symptoms were recorded for plants infected by the other isolates. Pathogens were reisolated from diseased plants. Disease symptoms were similar to those observed on plants collected in the field, and differed among pathogens. The Pythium isolates and P. irregulare caused tan discoloration and pruning of the entire root system, sometimes leaving only stubs along the taproot. Survival of clover plants infected by Pythium spp. and P. irregulare isolates averaged 79 and 83%, respectively. R. solani AG4 isolates caused internal crown discoloration, root lesions, and severe root rot, and plant survival was only 31%. F. proliferatum caused hyperelongation of the hypocotyl and stem, mild chlorosis, tan discoloration of roots, and 94% plant survival. Survival was 86% for control plants. P. ultimum, P. irregulare, R. solani AG4, and F. proliferatum are part of a larger disease complex occurring on arrowleaf clover in east Texas that also includes bean yellow mosaic virus (1) and possibly other root and crown rot diseases. This report is the first to identify some of the components of this disease complex impacting arrowleaf clover in Texas, and to demonstrate pathogenicity of each fungal pathogen individually. There are no arrowleaf clover cultivars currently known to have resistance to any of these pathogens. Reference: (1) I. J. Pemberton et al. Phytopathology 81:1001, 1991.