A methodology for risk analysis of plurivorous fungi in biological weed control: Sclerotinia sclerotiorum as a model

In New Zealand, research is being performed on the use of the ubiquitous plurivorous ascomycete Sclerotinia sclerotiorum, as a biocontrol agent for Cirsium arvense in pasture. As a consequence of the wide host range of this fungus, the proposed biocontrol may pose a risk to non-target arable plants. Crop disease risk is primarily due to the formation of sclerotia. These perennating bodies may directly infect susceptible crops sown at the biocontrol site in years after the biocontrol event. Additionally they may cause infection in neighbouring crops through the formation of apothecia from which airborne ascospores are dispersed. A methodology for a risk analysis is proposed, embodying both temporal and spatial processes, where the relative disease risk from biocontrol is defined as the ratio of added to naturally occurring inoculum.Exponential decay is proposed as a suitable model for the survival of sclerotia in soils beneath pastures, and data from an ongoing study are used in a preliminary parameterisation. The model is applied to published data on initial sclerotium population size and unpublished data on natural levels of soilborne sclerotia, to reveal how a withholding period, defined as the interval of time that must elapse for the ratio of added to natural inoculum to equal unity, may be calculated.For quantifying escape and aerial dispersal of ascospores from a treated pasture, three mechanistic models are proposed. Firstly, a simple one-layer model of escape, giving a provisional estimation of 81% of released ascospores escaping the pasture canopy, is discussed. Secondly, ascospore dispersal within and close to the biocontrol site (<30 m) is described by a model revealing abrupt decline in the atmospheric concentration of ascospores within short distances from the biocontrol site. Thirdly, for predicting ascospore concentrations at greater distances (>100 m), a Gaussian plume model is proposed. In a first approximation, these models are used in an example calculation to show how a hypothetical safety zone might be determined. A map of the province of Canterbury showing the locations of different agricultural activities and infestations of dense populations of C. arvense, revealed that potential biocontrol sites mostly do not border arable land and that a safety zone would therefore be feasible.The study presented here provides conceptual and mathematical frameworks for risk analysis of the use of plurivorous fungi in biological weed control, using S. sclerotiorum as an example. However, before a judgement may be made about the phytosanitory risks associated with the use of S. sclerotiorum to control C. arvense in pastures in Canterbury, the models developed in this paper must be parameterized using data collected under a wide range of conditions in the field.     

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.     

Forecasting the airborne spread of Mycosphaerella fijiensis, a cause of black Sigatoka disease on banana: estimations of numbers of perithecia and ascospores

Banana leaves showing different levels of black Sigatoka disease were collected from an unsprayed plantation in Costa Rica during two separate periods representing the wet to dry season transition (October 1993 – February 1994) and the dry to wet season transition (April – September 1995). Laboratory studies were used to investigate the relationship between the release of Mycosphaerella fijiensis ascospores and the amount of inoculum on banana plants showing different levels of infection, as assessed by leaf necrotic area. The number of perithecia present in the necrotic area was used as an indication of potential ascospore loads and was investigated as a series of regression equations. A series of rewetting and incubation regimes was used to investigate spore release under field conditions (21°C and 100% relative humidity in the early morning and 28°C, 60% relative humidity on days when it rained in mid-afternoon). Results suggest that rainfall, combined with a high temperature, may lead to peaks of ascospore release but without necessarily increasing overall numbers released over periods of up to 4 days and that a high level of spore release was less sensitive to changes in temperature once it had been initiated. The exact role of temperature in spore release is still unclear, however, as leaf samples kept at atypically low temperatures also released non-germinating ascospores. An average of 4.5 ascospores was released per perithecium. This does not resolve ambiguities in the literature regarding the number of ascospores present in each perithecium. A linear model relating the average ascospore numbers and necrotic area, using quick estimates of the amounts of necrotic area on the leaves of a random sample of plants across a plantation, is proposed, to give an indication of the relative amount of airborne inoculum potentially available between different plantations.     

Effect of timing of application Pseudomonas fluorescens in suppression Sclerotinia sclerotiorum, the causal agent of white mold in canola

Biological control of sclerotinia disease, as an important alternative to chemical control, has received considerable attention due to the lack of resistant varieties in most crop, and increasing concerns over fungicide resistance in population of Sclerotinia sclerotiorum, and fungicide residues in the environment. One biocontrol agent, Pseudomonas fluorescens PB-3, has been showed the antagonistic relationship between itself and S. sclerotiorum was investigated in this study. A petal infection technique was used to detect efficacy of timing of application strain PB-3 in the suppression of S. sclerotiorum on canola. Significant difference in disease severity (p<0.05) were found with respect to timing of ascospore applications in the control treatments (ascospores only). The superior competitive ability strain PB-3 was demonstrated by its complete suppression of disease severity when applied as a co-inoculation treatment or prior to ascospores inoculation. Analysis of effect of applying strain PB-3 after ascospores was indicated that treatment in which strain PB-3 was added to petals 48 or 24 h after ascospores, or when there were no bacteria present at all, had higher rates of disease progression. It would be appear that bacteria are able to significantly inhibit disease when applied before or even at the same time as ascospores. In a practical sense, this could mean that a field application of antagonist could be concurrent with infection by the pathogen.     

Influence of meteorological parameters on Leptosphaeria maculans and L. biglobosa spore release in central and eastern Poland

Leptosphaeria maculans and L. biglobosa are fungal pathogens able to cause allergic reactions in humans and infect plants of Brassica species. The rate of their development and subsequent spore release depend on weather conditions. The aim of this paper was to pinpoint the exact meteorological conditions triggering the release of L. maculans and L. biglobosa ascospores in central and eastern Poland. Multiple regressions indicated that the frequency and amount of rainfall over short periods were important in mediating spore release. The first ascospore event depended mainly on the number of rainy days during the first 10 days of July and the cumulative precipitation during July and September. The most important variables for maximum spore release were cumulative rainfall in the beginning of July and the end of September, as well as the number of days with precipitation events in the first 10 days of August. The results highlighted for the first time the importance of the days preceding the collection of oilseed rape plants from the field. Higher moisture content of senescing but still living stems play a crucial role in the early start of the ascospore season and the maximum release of ascospores. This was not yet considered to date.     

Airborne ascospores in Mérida (SW Spain) and the effect of rain and other meteorological parameters on their concentration

Airborne ascospores have been reported to be allergenic or plant pathogens, and their presence has traditionally been associated with rainfall events. The aim of the present study was to analyze the presence of airborne ascospores in relation to weather parameters in a town in SW Spain. A seven-day recording spore trap (Burkard) was used to sample the air over 2 years at 15 m above ground level on the terrace roof of the hospital in Mérida (SW Spain). Fungal spores were identified and counted by means of two longitudinal scans over the slides under ×1000 microscopy. A correlation analysis was made of the daily meteorological data and the airborne ascospore concentrations, and t-tests were used to compare data between the 2 years. Nineteen ascospore types were defined, including one-cell ascospores (Chaetomium, Diatrype, Helvella, Xylaria), tow-cell ascospores (Diaporthe, Mycosphaerella, Nectria, Venturia), transversally septate ascospores (Melanomma, Leptosphaeria, Paraphaeosphaeria, Sporormiella, Massaria), transversally and longitudinally septate ascospores (Pleospora), and ascospores within asci (Sordaria). Leptosphaeria consisted of a group of four types described according to the number of cells, hyaline grade, wall thickness, and ornamentation, and other ascospores comprised one last additional type. The average airborne ascospore concentration was 153 ascospores/m3. One-third each of this total were from the Leptosphaeria group, with an average 54 ascospores/m3, and the two-cell ascospores or Venturia-like group (Diaporthe, Mycosphaerella, Nectria, Venturia) with 51 ascospores/m3 on average. In third position was Pleospora with 27 ascospores/m3 on average. The month with highest concentration was September, with 238 ascospores/m3, and the lowest March, with 56 ascospores/m3. By seasons, autumn had the highest concentrations, followed by winter, spring, and summer. The maximum daily concentration reached was 3,371 ascospores/m3. Daily rainfall was significantly correlated with the ascospore types Diatrype, Mycosphaerella, Nectria, two subtypes of Leptosphaeria, and Pleospora. Relative humidity was positively correlated with those ascospore types and also with Diaporthe and Paraphaeosphaeria, and negatively with Chaetomium and Melanomma. The concentration was higher on rainy days than on days without rain for Pleospora, Leptosphaeria (3 subtypes), Diatrype, Diaporthe, Nectria, Mycosphaerella, and Paraphaeosphaeria. The daily temperatures were in general correlated with the same types as the relative humidity, but with the opposite sign. For the monthly data, there were no statistically significant differences between the 2 years studied.     

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.     

冬虫夏草子囊孢子的发育

经光学显微镜观察,冬虫夏草[Cordyceps sinensis(Berk.)Sacc.]子囊孢子的发育可分为三个阶段:(1)原子囊孢子期:孢子近球形,椭圆形,卵圆形或长圆形,4.8—10×3.6—6μm。(2)孢子伸长期:从孢子一端突出,伸长生长,出现分隔,隔细胞较长,30—37.5μm。(3)子囊孢子形成期:孢子继续伸长并不断出现分隔,隔间长6—12μm。一般仅有两个孢子成熟,其余多数原子囊孢子败育,不萌发或形成极纤细的丝状物。基于子囊孢子发育的过程,讨论了冬虫夏草及其近缘种的分类地位。     

Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ～50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.     

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.     

The release of elongated, sheathed ascospores from bottle-shaped asci in Dipodascus geniculatus

Yeasts use different mechanisms to release ascospores of different lengths from bottle-shaped asci. Round to oval-shaped ascospores are enveloped in oxylipin-coated compressible sheaths, enabling ascospores to slide past each other when they reach the narrowing ascus neck. However, more elongated ascospores do not contain sheaths, but are linked by means of oxylipin-coated interlocked hooked ridges on the surfaces of neighboring ascospores, thereby keeping them aligned while they are pushed towards the ascus tip by turgor pressure. In this study, we found elongated, oxylipin-coated sheathed ascospores in Dipodascus geniculatus that are released effectively from bottle-shaped asci without alignment. This is possible because the ascus neck and opening have a diameter that is the same as the length of the ascospore, thus allowing the ascospores to turn sideways without blocking the ascus when they are released. We found that increased concentrations of acetylsalicylic acid inhibit both ascospore release and 3-hydroxy oxylipin production in this yeast, thereby implicating this oxylipin in sexual reproduction.     

Apothecia and ascospores of Lobaria oregana and Lobaria pulmonaria investigated

Apothecia of Lobaria oregana and L. pulmonaria emerge in late spring and discharge ascospores throughout the year. Most populations have a few fertile thalli, although the proportion of fertile thalli usually is less than 25 percent. Ascospores fail to germinate in water or on water agar but do germinate on agar containing an adsorbant and either a sugar or the sugar-alcohol ribitol. It is postulated that the ascospores of these species contain an autoinhibitor that must be removed before germination. Widespread ascospore germination in the presence of an adsorbant in Peltigera aphthosa, P. membranacea, and Pseudocyphellaria anthraspis, as well as in Lobaria, suggest that this phenomenon might be widespread in the Peltigerales.     

Ascoma development and phylogeny of an apothecioid dothideomycete, Catinella olivacea

Catinella olivacea is a discomycetous fungus often found fruiting within cavities in rotting logs. Because this habitat would lack the air currents upon which discomycete species normally rely for the dispersal of their forcibly ejected ascospores, we suspected an alternative disseminative strategy might be employed by this species. An examination of the development of the discomycetous ascomata in pure culture, on wood blocks, and on agar showed that the epithecium was gelatinous at maturity and entrapped released ascospores in a slimy mass. We interpreted this as an adaptation for ascospore disperal by arthropods. Developmental data also showed that C. olivacea was unusual among other discomycetes in the Helotiales (Leotiomycetes). For example, the ascoma developed from a stromatic mass of meristematically dividing cells and involved the formation of a uniloculate cavity within a structure better considered an ascostroma than an incipient apothecium. Furthermore, the ascus had a prominent ocular chamber and released its ascospores through a broad, bivalvate slit. These features, along with phylogenetic analyses of large subunit and small subunit rDNA, indicated that this unusual apothecial fungus is, surprisingly, more closely affiliated with the Dothideomycetes than the Leotiomycetes.     

Effects of Weather Variables on Ascospore Discharge from Fusarium graminearum Perithecia

Fusarium graminearum is a predominant component of the Fusarium head blight (FHB) complex of small grain cereals. Ascosporic infection plays a relevant role in the spread of the disease. A 3-year study was conducted on ascospore discharge. To separate the effect of weather on discharge from the effect of weather on the production and maturation of ascospores in perithecia, discharge was quantified with a volumetric spore sampler placed near maize stalk residues bearing perithecia with mature ascospores; the residues therefore served as a continuous source of ascospores. Ascospores were discharged from perithecia on 70% of 154 days. Rain (R) and vapor pressure deficit (VPD) were the variables that most affected ascospore discharge, with 84% of total discharges occurring on days with R≥0.2 mm or VPD≤11 hPa, and with 70% of total ascospore discharge peaks (≥ 30 ascospores/m³ air per day) occurring on days with R≥0.2 mm and VPD≤6.35 hPa. An ROC analysis using these criteria for R and VPD provided True Positive Proportion (TPP) = 0.84 and True Negative Proportion (TNP) = 0.63 for occurrence of ascospore discharge, and TPP = 0.70 and TNP = 0.89 for occurrence of peaks. Globally, 68 ascospores (2.5% of the total ascospores sampled) were trapped on the 17 days when no ascospores were erroneously predicted. When a discharge occurred, the numbers of F. graminearum ascospores sampled were predicted by a multiple regression model with R² = 0.68. This model, which includes average and maximum temperature and VPD as predicting variables, slightly underestimated the real data and especially ascospore peaks. Numbers of ascospores in peaks were best predicted by wetness duration of the previous day, minimum temperature, and VPD, with R² = 0.71. These results will help refine the epidemiological models used as decision aids in FHB management programs.     

A fibrous inclusion body in developing ascospores ofAscobolus stercorarius

Summary Electron microscopic examination, using glutaraldehyde and osmium tetroxide as fixatives, has revealed the existence of a unique structure, the filamentous ascospore inclusion (FAI), in the sporoplasm of developing ascospores ofAscobolus stercorarius. The FAI, a highly ordered aggregate of 80–125 å diameter, rigid, filamentous tubules, is examined as to structure and origin; in particular, certain evidence suggesting these structures are the aggregated form of the intra-nuclear spindle is discussed. The functional role of the FAI is considered in relation to the current literature; a bifunctional role in the apothecium is considered in light of the occurrence of an additional class of filamentous structure in other differentiated elements of the ascocarp.     

Environmental Factors Influencing the Dispersal of Venturia inaequalis Ascospores in the Orchard Air

A 6-year study was carried out in an apple-growing region of North Italy by trapping airborne ascospores of Venturia inaequalis with a volumetric spore trap operated continuously during the ascospore season, with the aim of better defining the weather conditions that allow ascospores both to discharge and to disperse into the orchard air. A total of more than 60 ascospore trapping events occurred. Rain events were the only occurrences allowing ascospores to become airborne (a rain event is a period with measurable rainfall ≥0.2 mm/h – lasting one to several hours, uninterrupted or interrupted by a maximum of two dry hours); on the contrary, dew was always insufficient to allow ascospores to disperse into the air at a measurable rate, in the absence of rain. In some cases, rain events did not cause ascospore dispersal; this occurred when: (i) rain fell within 4–5 h after the beginning of a previous ascospore trapping; (ii) rain fell at night but the leaf litter dried rapidly; (iii) nightly rainfalls were followed by heavy dew deposition that persisted some hours after sunrise. Daytime rain events caused the instantaneous discharge and dispersal of mature ascospores so that they became airborne immediately; for night-time rainfall there was a delay, so that ascospores became airborne during the first 2 h after sunrise. This delay did not always occur, and consequently the ascospore trapping began in the dark, when: (i) the cumulative proportion of ascospores already trapped was greater than 80% of the total season's ascospores; (ii) more than one-third of the total season's ascospores was mature inside pseudothecia and ready to be discharged.     

Effects of temperature and rainfall on date of release of ascospores of Leptosphaeria maculans (phoma stem canker) from winter oilseed rape (Brassica napus) debris in the UK

Data from a controlled environment experiment investigating effects of temperature on maturation of Leptosphaeria maculans pseudothecia were used to derive equations describing the times until 30% or 50% of pseudothecia were mature as a function of temperature. A wetness sensor was developed to estimate the oilseed rape debris wetness and operated with debris exposed in natural conditions in 2000 and 2001. The maturation of L. maculans pseudothecia on debris and concentrations of airborne L. maculans ascospores were observed from 1999 to 2004. There were considerable differences between years, with the first mature pseudothecia observed in September in most years. There were linear relationships between the first date when 10% of maximum ascospore release was observed and the dates when 30% or 50% of pseudothecia were mature. By summing the daily temperature‐dependent rate of pseudothecial maturation for days after 1 August with rainfall >0.5 mm, the dates when 30% or 50% of pseudothecia were mature were predicted. There was good agreement between predicted and observed dates when 30% or 50% of pseudothecia were mature. These equations for predicting the timing of L. maculans ascospore release could be incorporated into schemes for forecasting, in autumn, the severity of phoma stem canker epidemics in the following spring/summer in the UK.     

Measuring summer patterns of ascospore release by saltmarsh fungi

One potentially important type of flux from standing-decaying marshgrass is the production and release of ascospores. The most extensive measurements of ascospore release from the principal marshgrass (Spartina alterniflora, smooth cordgrass) of saltmarshes of the eastern coastal United States involved an arbitrary, weeklong period of wet incubation of leaf-blade samples. We examined the possibility that shorter incubations would yield higher estimates of hourly rates of ascospore release, testing wet incubations of 3 to 71 h, using standing-decaying leaf blades of smooth cordgrass from low on living shoots and high on dead shoots. Incubations of 31 h appeared to be optimal. Species compositions of ascospores expelled from the two leaf types were distinctly different: high leaves yielded primarily aMycosphaerella species orPhaeosphaeria halima; low leaves yielded primarilyPhaeosphaeria spartinicola or theMycosphaerella species. All of these species consistently exhibited high coefficients of variation (>100%) for their mean rates of release of ascospores. Only theMycosphaerella species on high leaves gave evidence of a delayed onset of ascospore expulsion during incubation, and this evidence was equivocal. Grand mean rates of ascospore release forP. spartinicola and theMycosphaerella species were, respectively, 106 and 238 spores cm⁻² abaxial leaf area h⁻¹.     

The aerobiology of the ascospores

Atmospheric ascospores have been monitored using volumetric spore trap. Spore concentration data were analysed using Spearman's correlation. Our results show that the meteorological factor with the greatest effect on spore concentration was the duration of rain. Temperature increase strongly reduced the ascospore concentration; but the length of windless periods resulted in an increase in spore count. The only measurable effect wind perse actually had on spore count, was registered when a strong wind blew after a long windless period. We observed that the count of ascospores during wet weather could surpass the total concentration of dry conidia measured on a typical, highly polluted summer day. Using selected air samples to study the effect of storms, certain aspects of long-distance spore transport were elucidated. We describe here three main strategies for long-range ascospore transport, "splash-off", "secondary emission" and "sporematrix projectiles".