Fifty Years of Fungicide Development, Deployment, and Future Use.

Plant disease management has not significantly changed in the past 50 years, even as great strides have been made in the understanding of fungal biology and the etiology of plant disease. Issues of climate change, supply chain failures, war, political instability, and exotic invasives have created even more serious implications for world food and fiber security, and the stability of managed ecosystems, underscoring the urgency for reducing plant disease-related losses. Fungicides serve as the primary example of successful, widespread technology transfer, playing a central role in crop protection, reducing losses to both yield and postharvest spoilage. The crop protection industry has continued to improve upon previous fungicide chemistries, replacing active ingredients lost to resistance and newly understood environmental and human health risks, under an increasingly stricter regulatory environment. Despite decades of advances, plant disease management continues to be a constant challenge that will require an integrated approach, and fungicides will continue to be an essential part of this effort.

Medicaid Payment and Delivery System Reform: Early Insights from 10 Medicaid Expansion States.

Issue: Expanded Medicaid enrollment under the Affordable Care Act has heightened the importance of states' roles as principal purchasers of health care for low-income and medically vulnerable populations. Concurrently, the federal government has augmented states' purchasing tools. Goal: To examine the evolution of payment and delivery system reform in 10 ACA Medicaid expansion states. Methods: Analysis of state managed care policies, including a detailed review of purchasing documents as well as interviews with senior agency officials in 10 states. Findings and Conclusions: States have made health system reform a core element of their Medicaid expansions, with the aim of improving access, quality, efficiency, and population health. States have sought to incorporate evidence-based practice and payment strategies, with an emphasis on populations likely to benefit from improved care management and on better integration of treatment for physical and behavioral health problems. Seven of 10 are directly engaged in provider payment and delivery system reform. Agencies noted the importance of experienced provider networks in addressing complex health and social needs, along with managed care's role in quality improvement and payment reform. States embrace their roles as payers and health care innovators, identifying stability of both coverage and the underlying federal policy environment as key factors.

How Will Repealing the ACA Affect Medicaid? Impact on Health Care Coverage, Delivery, and Payment.

ISSUE: The Affordable Care Act enhanced Medicaid's role as a health care purchaser by expanding eligibility and broadening the range of tools and strategies available to states. All states have embraced delivery and payment reform as basic elements of their programs. GOAL: To examine the effects of reducing the size and scope of Medicaid under legislation to repeal the ACA. FINDINGS AND CONCLUSIONS: Were the ACA's Medicaid expansion to be eliminated and were federal Medicaid funding to experience major reductions through block grants or per capita caps, the effects on system transformation would be significant. Over 70 percent of Medicaid spending is driven by enrollment in a program that covers 74 million people; on a per capita basis Medicaid costs less than Medicare or commercial insurance. States would need to absorb major financial losses by reducing the number of people served, reducing the scope of services covered, introducing higher cost-sharing, or further reducing already low payments. Far from improving quality and efficiency, these changes would cause the number of uninsured to rise while depriving health care providers and health plans of the resources needed to care for patients and invest in the tools that are essential to system transformation

Mining the Gap: Assessing Leadership Needs to Improve 21st Century Plant Pathology.

Scientists and plant pathologists are trained in scientific knowledge and critical thinking as part of their career preparation process. However, the extensive training in science-related skills may come at a cost to "soft skills," the competencies needed for interpersonal skills, communication, management, and leadership. A survey of the American Phytopathological Society indicated that the vast majority of its members (91%) were in leadership and management roles. Despite this, a minority of survey participants felt that their scientific training had prepared them to lead (30%) or manage others (36%). Plant pathologists had received the most training in topics that were tied to science. Less common were critical topics such as resilience, entrepreneurship, visioning, and persuasion, and participants were likely to choose skills considered necessary for management, as opposed to leadership. While scientific training is the primary purpose of graduate training, the lack of leadership training and professional development represents a critical deficiency at a time when science is increasingly moving toward larger collaborative projects. Soft skills training options are available, but utilization of these resources needs to be encouraged. An increased emphasis on augmenting leadership and management skills is critical to prepare scientists for a competitive, dynamic, and increasingly collaborative science landscape.

First Report of Brown Rot (Monilinia fructicola) on the Dogwood, Cornelian Cherry (Cornus mas).

Cornelian cherries (Cornus mas), also called cornels, are members of the dogwood family (Cornaceae), and are not true cherries. Cornelian cherry is primarily grown as an edible landscape ornamental in the United States. Brown rot, caused by fungi in the genus Monilinia, is one of the most important diseases of stone fruit worldwide. In the United States, M. fructicola is the most commonly observed Monilina species, although M. fructigena and the European brown rot pathogen, M. laxa, may also infect stone fruit. M. fructigena is the only Monilinia species reported to infect cornelian cherry, but there is only a single report of it occurring in the United States (1,4). All three species have similar morphology and are commonly misidentified (1,3,4). In August of 2010 and 2013, in one location, brown rot was observed on fruit of the cornelian cherry cultivar Elegans. In both instances, only 'Elegans' fruit was infected while neighboring 'Sunrise' exhibited no symptoms in the field, and lesions did not appear to develop into shoot blight. In 2013, single-spore isolates from the diseased fruit were cultured on potato dextrose agar (PDA) incubated at 25°C for 5 days. Colony morphology was consistent with M. fructicola and was rapidly growing, gray, producing concentric rings, and developing smooth colony margins. Conidia were hyaline, 10 × 15 µm, and formed in branched, monilioid chains of varying lengths (1). Molecular-based species identification was performed on the 450-bp amplified ribosomal internal transcribed spacer (ITS) sequences, using primers ITS1 and ITS4. BLAST searches of the ITS sequences in GenBank showed the highest similarity (100%) with sequences of M. fructicola isolates from Italy (FJ411110), China (FJ515894), and Spain (EF207423). Pathogenicity was confirmed by inoculating surface-sterilized, mature 'Sunrise' fruit with mycelial plugs of the isolate identified with the ITS sequence. Mycelial plugs (3 mm in diameter) were removed from the periphery of a 5-day-old colony and placed upside down into five fruit that were wound-inoculated with a 3-mm cork borer, petiole hole-end inoculated, or unwounded but inoculated; control fruit for each treatment received sterile plugs of PDA as a control. All fruit was stored in a moist chamber for the duration of the experiment. Wound-inoculated fruit developed symptoms within 2 days; sporulating lesions developed within 5 days. Symptoms of infection via the petiole developed in 4 days; by day six, three of the five inoculated fruit were infected, and four of the five were infected by day eight. Unwounded, inoculated fruit showed symptoms on day six; three of the five fruit were infected by day eight. None of the control inoculations showed Monilinia infection. Pathogens were re-isolated from the inoculated fruit and confirmed to be M. fructicola on the basis of morphological characteristics. To our knowledge, this is the first fulfillment of Koch's postulates demonstrating that M. fructicola can infect cornelian cherry. A previous report by Höhnel in 1918 described infection by Lambertella corni-mas of a cornelian cherry in Austria; however, the taxonomic details presented are consistent with M. fructigena (2). References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) T. H. Harrison and A. F. El-Helaly. Brit. Mycol. Soc. Trans. 19:199, 1935. (3) C. R. Lane. EPPO Bulletin 32:489, 2002. (4) E. M. Sagasta. EPPO Bulletin 7:105, 1977.

First Report of the White Pine Blister Rust Fungus, Cronartium ribicola, on Ribes odoratum in Indiana.

Cronartium ribicola J. C. Fisch., causal agent of white pine blister rust (WPBR), is one of the most damaging pathogens of five-needle pines, forming aecial states on the trunk and branches and causing cankering, topkill, and branch dieback. Infection can predispose hosts to attack by other pests such as bark beetles, and can result in host mortality. Various species of Ribes, Pedicularis, and Castilleja are alternate hosts on which C. ribicola forms its uredinial and telial states during the mid-summer to fall. In an effort to mitigate the damage caused by white pine blister rust, the planting of ornamental species of Ribes, such as R. occidentalis, is prohibited in 14 states. Indiana currently has no restrictions on the planting of Ribes spp. Since 2010, a Cronartium sp. has been observed producing uredinia and telia on R. odoratum 'Crandall' H.L. Wendl. leaves in an urban environment in West Lafayette, Indiana. Symptoms include yellow-orange lesions on the leaf upper surface with uredinia on the underside. These persist from late summer until leaf drop. Telia were collected in 2011 to establish the identity of the causal agent using morphological and molecular analyses. Morphological comparisons between this specimen and other Cronartium species were made using Arthur (2). Filiform telial columns ranged from 0.5 to 1.5 mm in length. Teliospores were cylindrical to sub-ventricose, truncate on either end with one end generally tapering more than the other, and measured 9.0 to 18.6 × 37.2 to 60.0 µm (average 11.9 × 47.4 µm from 30 spores across 4 leaves). These teliospore measurements overlap those of C. ribicola and C. occidentale, but are more consistent with C. ribicola, in which the spores are wider and longer (8 to 12 × 30 to 60 µm) than in C. occidentale (9 to 10 × 27 to 56 µm). For molecular analyses, two nuclear ribosomal loci were sequenced: the internal transcribed spacer regions 1, 2, and 5.8S (ITS) and the 5' end of the large subunit (28S) (1). The ITS sequence was 665 bp long (KF387533) and the 28S was 892 bp (KC876675). These sequences were queried to GenBank using a BLASTn search. The 28S shared 99% identity (891/892 bp) and the ITS shared 100% identity (663/663 bp) to other published C. ribicola sequences with no close matches to any other species with either locus. Both morphological and molecular methods indicate this species to be C ribicola, making this a first report of white pine blister rust on R. odoratum in Indiana. This fungus has been observed previously on R. odoratum in the northeastern United States (Connecticut, Massachusetts, Rhode Island, Vermont, and New Hampshire), the Rockies (Colorado), northwestern United States (Washington), and Canada (3). In Indiana, C. ribicola has also been reported on R. cysnobati. There are no other reports of this fungus on any other host within the state. However, the aecial host, Pinus strobus, does grow within the state, and within West Lafayette. To our knowledge, WPBR has only been observed (not reported) once in Indiana in the past 30 years (Paul Pecknold, personal communication). Further monitoring of C. ribicola hosts is needed in Indiana to determine the extent of the disease. The specimen has been vouchered in the Arthur Herbarium (PUR N6734). References: (1) M. C. Aime. Mycoscience 47:112. 2006. (2) J. F. Arthur. Manual of the Rusts in United States and Canada. Purdue Research Foundation, 1934. (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/ April 23, 2013.

A Survey of Phytophthora spp. in Midwest Nurseries, Greenhouses, and Landscapes.

A survey of nurseries, greenhouses, and landscapes was conducted from 2006 to 2008 in order to determine the prevalence and diversity of Phytophthora spp. From sites in Iowa, Michigan, Ohio, and, predominantly, Indiana, 121 Phytophthora isolates were obtained from 1,657 host samples spanning 32 host genera. Based on sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA, 11 Phytophthora spp. and two hybrid species were identified. A majority of the isolates were P. citricola (35.9%) or P. citrophthora (27.4%). Six isolates were confirmed as hybrids (four of P. cactorum × hedraiandra and two of P. nicotianae × cactorum) by cloning and sequencing the ITS region. Three P. cactorum × hedraiandra isolates were obtained from the same site, from three Rhododendron spp., which are known hosts to the parental species. The fourth isolate, however, was recovered out of a different location in a Dicentra sp., which is not a known host to either parental species, suggesting an expansion of host range of the hybrid isolate as compared with either parental species.

A system for studying genetic changes in Candida albicans during infection.

Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the C. albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of C. albicans from a commensal to a pathogenic organism.

A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking.

The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome), and YAC (yeast artificial chromosome).

Single-copy IMH3 allele is sufficient to confer resistance to mycophenolic acid in Candida albicans and to mediate transformation of clinical Candida species.

Parasexual genetic analysis of Candida albicans utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the IMH3(r) gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213-218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331-2338, 1997) have shown that the wild-type IMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the IMH3(r) gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis. We also identified the presence of autonomously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a phi-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3(r) allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

Fine-resolution physical mapping of genomic diversity in Candida albicans.

It has been suggested that Candida albicans, a diploid asexual fungus, achieves genetic diversity by genomic rearrangement. This important human pathogen may provide a system in which to analyze alternate routes to genomic diversity. C. albicans has a highly variable karyotype; its chromosomes contain a middle repeated DNA sequence called the Major Repeat Sequence (MRS), composed of subrepeats HOK, RPS, and RB2. RPS is tandemly repeated while the other subrepeats occur once in each MRS. Chromosome 7, the smallest of the eight chromosomes, has been previously mapped. The complete physical map of this chromosome was used to analyze chromosome 7 diversity in six strains, including two well-characterized laboratory strains (1006 and WO-1) and four clinical ones. We found four types of events to explain the genomic diversity: 1) Chromosome length polymorphism (CLP) results from expansion and contraction of the RPS; 2) reciprocal translocation occurs at the MRS loci; 3) chromosomal deletion; and (4) trisomy of individual chromosomes. These four phenomena play an important role in generating genomic diversity in C. albicans.

Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae alpha-factor.

Magnaporthe grisea is a fungal pathogen with two mating types, MAT1-1 and MAT1-2, that forms a specialized cell necessary for pathogenesis, the appressorium. Saccharomyces cerevisiae alpha-factor pheromone blocked appressorium formation in a mating type-specific manner and protected plants from infection by MAT1-2 strains. Experiments with alpha-factor analogs suggest that the observed activity is due to a specific interaction of alpha-factor with an M. grisea receptor. Culture filtrates of a MAT1-1 strain contained an activity that inhibited appressorium formation of mating type MAT1-2 strains. These findings provide evidence that a pheromone response pathway exists in M. grisea that can be exploited for plant protection.

MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition.

Upon encountering a leaf surface, emergent germ tubes from conidia of the rice blast fungus, Magnaporthe grisea, form infection structures called appressoria that allow direct penetration of plant cells. The MPG1 gene encodes a fungal hydrophobin of M. grisea that is expressed during development of aerial hyphae, conidia, and appressoria. Deletion of MPG1 reduces the efficiency of appressorium formation. We found that yeast extract repressed MPG1 expression in vitro and inhibited appressorium development of the rice pathogen, strain Guy11. Appressorium formation of mpg1 mutants is rescued in trans by coinoculation with wild-type cells. MPG1 is required for efficient induction of appressoria in response to a host surface or highly hydrophobic artificial substrates. However, we identified several artificial substrates that can support efficient appressorium formation of mpg1 strains. This finding suggests that Mpg1p is not specifically required for appressorium formation, but is involved in the interaction with, and recognition of, the host surface. Additionally, a time window of competence to form appressoria was identified; the decision to form appressoria occurs approximately 6 to 8 h following conidial germination. After this critical time, cells are no longer able to form appressoria in response to inductive cues. These studies indicate that MPG1 hydrophobin is required for host recognition and that it acts as a morphogenetic signal for cellular differentiation.

Hyperosmolar cold storage kidney preservative solution.

A hyperosmolar kidney preservative solution which can maintain kidneys from experimental animals viable for up to 72 hours is described. Using the criterion of a one-month failure rate, the cold storage preservation method was found to be superior to machine preservation methods. Sachs' solution was found to be superior to all other cold storage solutions. The most important aspect of the hyperosmolar kidney preservative solution appears to be its ability to maintain normal intracellular electrolyte composition and to prevent cellular damage due to swelling. The present formula requires that the basic solution and a magnesium chloride additive solution be prepared separately and combined before use. A stable combined solution is proposed which can be sterilized by membrane filtration.