Sclerotinia sclerotiorum is a fungal plant pathogen and the causal agent of lettuce drop, an economically important disease of California lettuce. The structure of the S. sclerotiorum mating type locus MAT has previously been reported and consists of two idiomorphs that are fused end-to-end as in other homothallics. We investigated the diversity of S. sclerotiorum MAT using a total of 283 isolates from multiple hosts and locations, and identified a novel MAT allele that differed by a 3.6-kb inversion and was designated Inv+, as opposed to the previously known S. sclerotiorum MAT that lacked the inversion and was Inv-. The inversion affected three of the four MAT genes: MAT1-2-1 and MAT1-2-4 were inverted and MAT1-1-1 was truncated at the 3’-end. Expression of MAT genes differed between Inv+ and Inv- isolates. In Inv+ isolates, only one of the three MAT1-2-1 transcript variants of Inv- isolates was detected, and the alpha1 domain of Inv+ MAT1-1-1 transcripts was truncated. Both Inv- and Inv+ isolates were self-fertile, and the inversion segregated in a 1∶1 ratio regardless of whether the parent was Inv- or Inv+. This suggested the involvement of a highly regulated process in maintaining equal proportions of Inv- and Inv+, likely associated with the sexual state. The MAT inversion region, defined as the 3.6-kb MAT inversion in Inv+ isolates and the homologous region of Inv- isolates, was flanked by a 250-bp inverted repeat on either side. The 250-bp inverted repeat was a partial MAT1-1-1 that through mediation of loop formation and crossing over, may be involved in the inversion process. Inv+ isolates were widespread, and in California and Nebraska constituted half of the isolates examined. We speculate that a similar inversion region may be involved in mating type switching in the filamentous ascomycetes Chromocrea spinulosa, Sclerotinia trifoliorum and in certain Ceratocystis species.

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection.

Summary Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia‐lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA‐responsive pathogenesis‐related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription‐coupled quantitative PCR and RNA‐Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up‐regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV‐infected ZmPAL‐silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA‐mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV‐induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

The epidemiology of lettuce downy mildew has been investigated extensively in coastal California. However, the spatial patterns of the disease and the distance that Bremia lactucae spores can be transported have not been determined. During 1995 to 1998, we conducted several field- and valley-scale surveys to determine spatial patterns of this disease in the Salinas valley. Geostatistical analyses of the survey data at both scales showed that the influence range of downy mildew incidence at one location on incidence at other locations was between 80 and 3,000 m. A linear relationship was detected between semivariance and lag distance at the field scale, although no single statistical model could fit the semi-variograms at the valley scale. Spatial interpolation by the inverse distance weighting method with a power of 2 resulted in plausible estimates of incidence throughout the valley. Cluster analysis in geographic information systems on the interpolated disease incidence from different dates demonstrated that the Salinas valley could be divided into two areas, north and south of Salinas City, with high and low disease pressure, respectively. Seasonal and spatial trends along the valley suggested that the distinction between the downy mildew conducive and nonconducive areas might be determined by environmental factors.

Extensive studies have been conducted on the carpogenic germination of Sclerotinia sclerotiorum, but carpogenic germination in S. minor has not been studied adequately. It remains unclear why apothecia of this pathogen have seldom been observed in nature. In this study, a new method was developed to produce apothecia in the absence of soil or sand, and carpogenic germination without preconditioning was recorded for 95 of the 96 S. sclerotiorum isolates tested. Carpogenic germination of the two species was compared under a variety of temperature, soil moisture, burial depths, and short periods of high temperature and low soil moisture. The optimal temperatures for rapid germination and for maximum germination rates were both lower for S. minor than for S. sclerotiorum. The temperature range for carpogenic germination was also narrower for S. minor than for S. sclerotiorum. A 5-day period at 30 degrees C, either starting on the 10th or 20th day of incubation, did not significantly affect carpogenic germination of S. sclerotiorum. For both S. minor and S. sclerotiorum, the percentage of carpogenically germinated sclerotia increased as soil water potential increased from -0.3 to -0.01 MPa. In the greenhouse, a 10- or 20-day dry period completely arrested carpogenic germination of S. sclerotiorum, and new apothecia appeared after an interval of 35 days following rewetting, similar to the initial carpogenic germination regardless of when the dry period was imposed. In naturally infested fields, the number of sclerotia in 100 cc of soil decreased as depth increased from 0 to 10 cm before tillage, but became uniform between 0 and 10 cm after conventional tillage for both species. Most apothecia of S. minor were, however, produced from sclerotia located at a depth shallower than 0.5 cm while some apothecia of S. sclerotiorum were produced from sclerotia located as deep as 4 to 5 cm. These results provide the much needed information to assess the epidemiological roles of inoculum from sexual reproduction in diseases caused by the two Sclerotinia species in different geographical regions. However, more studies on effects of shorter and incompletely dry periods are still needed to predict production of apothecia of S. sclerotiorum in commercial fields under fluctuating soil temperature and moisture.

Experiments to identify the factors affecting survival of Bremia lactucae sporangia after deposition on lettuce leaves were conducted in growth chambers and outdoors under ambient conditions. Lettuce seedlings at the four-leaf stage were inoculated with B. lactucae sporangia under dry conditions. Sporangia deposited on lettuce seedlings were incubated at different temperature and relative humidity (RH) combinations, exposed to 100, 50, 25, and 0% sunlight in the second experiment, and exposed to different artificial lights in wavelength ranges of UVA (315 to 400 nm), UVB (280 to 315 nm), or fluorescent light in the third experiment. After exposure for 0 to 48 h in the first experiment and 0 to 12 h in the second and third experiments, seedlings in two pots were sampled for each treatment, and sporangia were washed from 15 leaves excised from the sampled seedlings. Germination of sporangia was determined in water after incubation in the dark at 15 degrees C for 24 h. The sampled seedlings with remaining leaves were first transferred to optimal conditions for infection (24 h), for the development of downy mildew, and then assessed for disease after 9 days. Sporangia survived much longer at 23 degrees C (>12 h) than at 31 degrees C (2 to 5 h), regardless of RH (33 to 76%). Germination percentage was significantly reduced after exposure to 50 and 100% sunlight. UVB significantly reduced sporangium viability, while fluorescent light and UVA had no effect relative to incubation in the dark. Infection of seedlings followed a pattern similar to germination of sporangia. Solar radiation is the dominant factor determining survival of B. lactucae sporangia, while temperature and RH have small, insignificant effects in coastal areas of California. This suggests that infections by sporangia that survived a day are probable only on cloudy days or on leaves that are highly shaded.

To understand the geographical distribution of lettuce drop incidence and the structure of Sclerotinia minor and S. sclerotiorum populations, commercial lettuce fields were surveyed in the Salinas, San Joaquin, and Santa Maria Valleys in California. Lettuce drop incidence, pathogen species, and mycelial compatibility groups (MCGs) were determined and analyzed using geostatistic and geographical information system tools. Lettuce drop incidence was lowest in the San Joaquin Valley, and not significantly different between the other two valleys. Semivariogram analysis revealed that lettuce drop incidence was not spatially correlated between different fields in the Salinas Valley, suggesting negligible fieldto-field spread or influence of inoculum in one field on other fields. Lettuce drop incidence was significantly lower in fields with a surface drip system than in fields with furrow or sprinkler irrigation systems, suggesting that the surface drip system can be a potential management measure for reducing lettuce drop. In the San Joaquin Valley, S. sclerotiorum was the prevalent species, causing drop in 63.5% of the fields, whereas S. minor also was identified in 25.4% of the fields. In contrast, in the Salinas Valley, S. minor was the dominant species (76.1%) whereas S sclerotiorum only observed in only 13.6% fields, in which only a few plants were infected by S. sclerotiorum. In the Santa Maria Valley, both species frequently were identified, with S. minor being slightly more common. Although many MCGs were identified in S. minor, most of them consisted of only one or two isolates. In all, ≈91.4% of the isolates belonged to four MCGs. Among them, MCG-1 was the most prevalent group in all three valleys, accounting for 49.8% of total isolates. It was distributed all over the surveyed areas, whereas other MCGs were distributed more or less locally. Populations of S. sclerotiorum exhibited greater diversity, with 89 isolates collected from the Salinas and San Joaquin Valleys belonging to 37 different MCGs. Among them, the most recurrent MCG-A contained 16 isolates, and 30 MCGs contained only 1 isolate each. Many MCGs occurred within only one or a part of the two valleys. Potential reasons for this abundant diversity are discussed.

The identities of Sclerotinia isolates obtained from chickpea plants showing stem and crown rot were determined using morphological characteristics, variations in group I introns, and internal transcribed spacer (ITS) sequences. Isolates could be separated into two groups based on growth rates at 22°C, fast growing (about 40 mm per day) versus slow growing (about 20 mm per day). All fast-growing isolates induced stronger color change of a pH-indicating medium than did slow-growing isolates at 22°C. The slow-growing isolates contained at least one group I intron in the nuclear small subunit rDNA, whereas all fast-growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow-growing isolates were identical to sequences of Sclerotinia trifoliorum. Those of the fast-growing isolates were identical to sequences of S. sclerotiorum. Finally, the slow-growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast-growing isolates showed no ascospore dimorphism. Isolates of both species were pathogenic on chickpea and caused symptoms similar to those observed in the field. This study not only associated the differences between S. sclerotiorum and S. trifoliorum in growth rates, group I introns, ITS sequences, and ascospore morphology, but also represented the first report that S. trifoliorum causes stem and crown rot of chickpea in North America.