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The role of resting spores in the survival of the mite-pathogenic fungus Neozygites floridana from Mononychellus tanajoa during dry periods in Brazil
Journal of Invertebrate Pathology 81 (2002) 148–157 www.academicpress.com
The role of resting spores in the survival of the mite-pathogenic fungus Neozygites floridana from Mononychellus tanajoa during dry periods in Brazil Sam L. Elliot,a,* John D. Mumford,b and Gilberto J. de Moraesc a
NERC Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK Environmental Science and Technology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK Departamento de Entomologia, Fitopatologia e Zoologia Agrıcola, Escola Superior de Agricultura ‘‘Luiz de Queiroz,’’ ESALQ/USP 13418-900, Piracicaba, SP, Brazil b
c
Received 15 January 2002; accepted 10 June 2002
Abstract Survival of pathogens during long periods of unfavorable conditions can be critical to their ecology and to their use in biological control. In northeastern Brazil, the mite pathogen Neozygites floridana must survive hot and dry conditions between wet seasons when it infects the cassava green mite Mononychellus tanajoa. We report on large numbers of mite cadavers bearing resting spores towards the end of epizootics in mid-1995. High within-leaf variability indicated that local factors may be important in determining resting spore formation. These spores remain in the host cadaver on a leaf until the cadaver breaks up, whereupon the spores fall freely to the soil, there to remain dormant. Laboratory simulation of field conditions led to ca. 25% of mycosed individuals bearing resting spores. Mummies (without resting spores) kept in hot and dry conditions showed little or no viability within 2 months, implying no role for survival over extended dry periods. It is proposed that resting spores form the principal means by which this pathogen survives the dry season in the study area. This has implications for its introduction to new areas in classical biological control. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Neozygites floridana; Cassava green mite; Mononychellus tanajoa; Resting spores; Entomophthorales; Tetranychidae; Overwintering; Biological control; Mummies
1. Introduction Although biologists working on temperate communities often ascribe little seasonality to tropical insect– pathogen systems (e.g., Briggs and Godfray, 1996), dry seasons near the equator can impose as much environmental variability on a pathogen population as do temperate winters. This is especially true of fungi but some are remarkably well adapted to surviving hot and dry conditions for long periods (e.g., Thomas et al., 1996). The survival of insect- or mite-pathogens for extended periods outside the host is thought to select for virulence and to maintain their effectiveness as biological control agents (Hochberg, 1989; Gandon, 1998). *
Corresponding author. Fax: +44-1344-873173. E-mail address: [email protected] (S.L. Elliot).
It is important to understand this aspect of pathogen ecology, and this is exemplified in the case of Neozygites floridana (Weiser & Muma) Remaudiere & Keller (Entomophthorales: Neozygitaceae) infecting the cassava green mite Mononychellus tanajoa (Bondar) (Acari: Tetranychidae). This fungus is of interest for biological control of this pest mite in both the Neotropics where they are endemic, and in Africa where the mite is exotic (Elliot et al., 2000; van der Geest et al., 2000; Yaninek et al., 1996). Epizootics of N. floridana in northeastern Brazil are sporadic, characterized by long periods of inactivity coinciding with dry seasons (Elliot et al., 2000). While N. floridana is capable of producing resting spores, these structures occur rarely and at low levels during epizootics (Delalibera et al., 1992, 1999; Elliot et al., 2000, 2002; Yaninek et al., 1996), raising the question of how this fungus survives dry seasons,
0022-2011/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 2 2 - 2 0 1 1 ( 0 2 ) 0 0 1 9 2 - 1
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especially in semi-arid environments such as northeastern Brazil. The production of thick-walled resting spores (sexual zygospores or asexual azygospores) by entomophthoralean fungi can be seen as a specific adaptation to survival in inhospitable conditions or in the absence of potential hosts, although additional benefits may accrue through the generation of novel genotypes in the sexual zygospores. However, other mechanisms of survival are potentially available to these fungi. Principal among these is the survival in mummified cadavers of the host (‘‘mummies’’). Note that we do not use the term ‘‘mummy’’ for resting spore-filled cadavers as we saw no evidence of mummification of the host cadaver. Other forms include the spores normally involved in the cycling of the pathogen, primary or secondary conidia, or as active infections in a different population of the host (Brandenburg and Kennedy, 1981) or in another host species. Mummies of N. floridana are actually sclerotia formed from mummified host cadavers and contain hyphal bodies of the fungus. These structures prevent desiccation of the fungus for short periods in the course of an epizootic. They have been implicated in the survival during harsh winters in temperate climes of N. floridana from the two spotted spider mite Tetranychus urticae Koch (Brandenburg and Kennedy, 1981). An isolate from M. tanajoa in Piritiba remained viable for up to 16 months at 5% relative humidity and 4 °C, but this was reduced to 6–7 months at the same humidity at 24 °C (Oduor et al., 1995). Although this latter study demonstrated the potential of the fungus to survive extended periods as mummies, it did not very closely reflect natural field conditions, where daily temperatures often exceed 35 °C and are much higher in the surface layers of the soil (SLE pers. obs.). The two spore forms produced in the asexual cycle, primary conidia and capilliconidia, are unsuitable for long-term persistence in dry conditions as they can only survive for short periods at high temperatures and low humidities (Oduor et al., 1995, 1996). Survival at a low prevalence in the host population seems unlikely as it was not detected during several years of monitoring of M. tanajoa populations in Piritiba (Elliot et al., 2000). Similarly, survival in alternative hosts is unlikely as the host mite is almost exclusively found on cassava (de Moraes et al., 1993) and this strain of the pathogen is very specific to M. tanajoa (de Moraes and Delalibera, 1992; Hountondji et al., 2002b; Leite et al., 2000). For these reasons, we suspect that N. floridana from M. tanajoa survives dry seasons as resting spores, despite their rarity. We report on finding mites filled with resting spores in mid-1995, describing their appearance and occurrence, and their immediate fate in the field. We also report on a preliminary and successful attempt to produce resting spores in vivo in the laboratory. To
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assess the potential of N. floridana to survive as mummies, we conducted a test of their long-term viability in the laboratory at a range of temperatures and saturation deficits intended to reflect field conditions, as well as on cassava stems and in soil in the field.
2. Materials and methods All fieldwork was carried out in growersÕ cassava fields in Piritiba, central Bahia, northeastern Brazil. All field-collected material was taken to the laboratory and examined within a few hours under a dissection microscope. 2.1. Resting spores on plants Following the initial detection of resting spore-filled M. tanajoa cadavers on cassava leaves in the study area on 25 June 1995, several thousand leaves were collected from more than 30 fields in the region, as well as five entire cassava stems (ca. 30 leaves per stem). Cadavers were examined. Mummies are easily recognizable as light brown. Any darker cadavers found on the leaf were removed and squashed on filter paper or microscope slides, or were mounted for subsequent microscopic investigation. Systematic observations were made in two cassava fields of local variety grouping Olho Roxo. Fields ‘‘1’’ and ‘‘2’’ were 50 m apart and were 4 and 18 months old and 1.5 and 2.0 m high, respectively. From 29 June to 19 September 1995, samples of 15–30 apical and median leaves were taken at irregular intervals from each field. The number of live M. tanajoa per leaf and the feeding injury from these mites was estimated. Total counts were made of the mummies and resting spore-filled cadavers per leaf. To obtain rough estimates of mite densities, these were estimated as scores for each leaf and then converted to previously calibrated means (estimates of 0, 1–25, 26–200, >200 mites per leaf receive scores of 0, 1, 2, 3 and conversion factors of 0, 10, 110, 350, respectively; Yaninek et al., 1989). Estimates of leaf injury were based on a visual estimate of percentage chlorosis where 0, no visible injury; 1 ¼ 1–25%; 2 ¼ 26–50%; 3 ¼ 51–75%; 4 ¼ 76–100%. These estimates were quick and were sufficient to characterize mite densities and feeding injury. The irregular timing of samples was due to lengthy counts (particularly of spore-filled cadavers) and limited time. To indicate how much local factors determined the formation of resting spores or mummies, variance:mean ratios were calculated from the counts per leaf (squareroot transformed) of resting spore-filled cadavers and mummies for each sample where both were found. Their variances were compared using F tests.
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A multiple regression analysis was conducted in GLIM version 4 (Royal Statistical Society, 1993; Crawley, 1993) to test whether some of the variation in the proportion of killed mites that formed resting spores could be explained by the measured parameters. Data for all individual leaves collected from 29 June to 20 July were put into one data set. The number of mites per leaf containing resting spores was taken as the explanatory variable with the total number of Neozygites-killed mites as the binomial denominator. A binomial error structure was incorporated in the modelling, with a logit link function. The independent variables were: sample date, field, stratum (apical or median), active mite densities, feeding injury and the numbers of Neozygites-killed mites. A maximal model was fitted to the data and then was simplified by a combination of backward and forward stepwise elimination of parameters as described by Elliot et al. (2000), but using v2 tests rather than F tests (Crawley, 1993). When parameters were removed from the model, their transformed equivalents were tested by inclusion where relevant. These were squares for mite densities and feeding injury, as these were scores, and square root transformations for dead mite numbers as these were counts. The data in the minimal model were highly over-dispersed (clumping of data points at the tails of frequency distributions). To account for this in the analysis, the scale parameter was adjusted according to Crawley (1993, p. 278) and the significance of terms was tested through F tests of the change in deviance upon their removal. 2.2. The immediate fate of resting spores To determine if resting spore-filled cadavers fell from leaves to the ground, traps were made from round plastic bowls of 0:18 m2 area and 20 cm height. Each had a row of three adjacent holes of 1 cm diameter bored in the base and covered with mite-proof gauze. Five of these traps were placed in each of the two fields, below cassava plant apices and slightly tilted to allow the drainage of rainwater. The traps were examined daily at the outset, then at intervals of up to eight days from 14 July (at this stage it was apparent that very little material would be lost from the traps as they were protected from wind and rain-splash by the cassava canopy). Material caught in the traps was removed with a hair brush and placed in individual vials containing 70% alcohol. The vials were taken to the field station and the contents filtered using filter paper. Examining the papers under the dissecting microscope, all objects resembling mites were collected and mounted in AmmanÕs Blue/HoyerÕs medium mixture for microscopic examination. To determine if resting spore-filled cadavers remained on fallen leaves, fallen and dried leaves were collected from cassava fields where resting spores had been found in abundance. Approximately 10 kg of leaf litter were
collected from three fields in the dry season following the epizootic. These were stored dry and examined for evidence of Neozygites-killed mites. 2.3. Resting spore formation in the laboratory To attempt to observe resting spore formation in the laboratory, the conditions under which they were found in the field were simulated as closely as possible. The conditions were: night temperature minima approximately 15 °C with relative humidity maxima approaching 100%; daytime temperature maxima approximately 25 °C with relative humidity minima 30–50%; photoperiod 11L:13D; overcast conditions; leaves with 30–50% chlorosis due to mite feeding damage; evidence of recently high mite and pathogen levels (exuviae, faeces, and sporulated cadavers), principally nymph cadavers bearing resting spores. Thus, 20 discs of 2 cm diameter were cut from field-collected cassava leaves bearing 30– 50% chlorosis from mite feeding. These were placed on water-soaked foam mats in petri dishes. Four laboratory-produced mummies were placed on each disc and left for 48 h in the dark at 20 °C, 90% RH. Thirty M. tanajoa nymphs were placed on each disc and these were then kept in a regime of 11 h light, 25 °C, 50% RH, and 13 h dark, 15 °C, 90% RH. The discs were observed daily and any dead mites were removed and mounted for examination under the microscope. This procedure was repeated once. Routine in vivo production of mummies was conducted concurrently using the same source of fungus and of mites and the same procedure to produce inoculum but using undamaged leaf material, young adult female mites, and a post-inoculation regime of continuous light, 25 °C, and approximately 70% RH. 2.4. Test of mummy viability Mummies were produced for this experiment using the routine in vivo procedure described above. This was timed so that all mummies used in the experiment were produced within five days of one another. Every day, new mummies were collected and either stored at 25 °C over pure glycerol (for the field experiment) or placed directly in the relevant storage vials (for the laboratory experiment; see below). To test the survival of mummies under a range of conditions in the laboratory, nine treatments combining three temperature and saturation deficit regimes were used, in addition to a control (8 °C, over pure glycerol) (Table 1). For each replicate, 10 ml of the appropriate solution of glycerol (Carson, 1931; Johnson, 1940) was placed in a black plastic film canister. Cotton wool was placed in the tube, leaving the base soaked in the glycerol and a dry cotton wool bed above, upon which to store the mummies. Fifteen mummies were placed in each vial, with 10 vials per
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Table 1 Temperature and saturation deficit treatments used in the laboratory experiment to test viability of N. floridana mummies over extended periods Temperature
Saturation deficit (mm Hg)
Relative humidity (%)
Glycerol concentration used (%)
25 °C
6 13 20
75 45 16
54 74 94
30 °C
6 13 20
81 59 37
41 63 80
35 °C
6 13 20
86 69 53
34 48 68
The corresponding relative humidities and the glycerol concentrations required to achieve these are shown.
treatment. Temperature and relative humidity were monitored in larger plastic boxes. For the field treatments, twenty mummies were placed in small white nylon bags, each within a larger, more porous, brown nylon bag. On 10 September, 1996, the bags were placed in a cassava field, either in the upper layer of soil, or at the base of a cassava plant. These were lightly covered with earth or tied to a plant stem, respectively. For weekly estimates of soil moisture, sub-samples of the top 1 cm of soil from the bases of five cassava plants were taken from three separate areas in the field. The samples were weighed, dried for 48 h at 105 °C, and re-weighed. The difference in weights was divided by the wet weight and multiplied by 100 to estimate percentage moisture. Means were taken of the three samples. After 37, 62, and 73 days, one unopened canister or bag was collected from each treatment (laboratory and field) and from the control. Six of the twenty mummies were taken at random from each, due to time constraints in assessing sporulation, and were placed on clean microscope slides in a dark water-saturated environment at 20 °C for 48 h. These were subsequently mounted with AmmanÕs Blue and examined under the microscope. The presence or absence of hyphal bodies, conidiophores and primary conidia was recorded for each mummy. A total count of primary conidia and capilliconidia was made on each slide.
3. Results 3.1. Resting spore-filled mites on plants—general observations Daily monitoring of the region of Piritiba led to the detection of epizootics of N. floridana on 25 June, and subsequently of resting spore-filled cadavers. These were very difficult to distinguish from other M. tanajoa which had also died and turned black. This was particularly true of mummies which had already sporulated as these
were rapidly colonised by saprophytic fungi. Many hours of handling material were needed to learn to distinguish between these cadavers with confidence and about a third of the resting spore-filled cadavers required microscopic examination for confirmation. Although full counts could take up to an hour for a leaf, many leaves could be examined very quickly. The maximum number of resting spore-filled cadavers found on a single leaf was 210. They were found in the later stages of epizootics in all the fields examined (>30) and were initially found in very low numbers of adult female mites. Subsequently, they were mostly observed at higher numbers predominantly in nymphs (there were incidentally very few adults left alive at this stage). None were found in larvae. This pattern appeared to reflect a wave of initially high mortality and population reduction among mature mites, followed by infection and mortality among the next generation as these matured from eggs or larvae. There was no visually apparent difference in the within-leaf distribution of mummies versus spore-filled cadavers, except that the latter were commonly found on the very edges of the leaf lobe borders where these were curled, and also on the leaf petiole where it joins the body of the leaf. Indeed, this served as a rapid indicator of the presence of resting spores in the field using a hand lens. Recently killed mites filled with resting spores were brown to grey, contrasting with the brown to yellow observed with recently killed and mummifying mites filled with hyphal bodies. A few live mites were found bearing clear immature resting spores. No dead mites were found with both resting spores and conidiophores or primary conidia—i.e., the sexual and asexual cycle seemed to be mutually exclusive within an individual host. Cadavers ultimately became pitch black, with colorless or partially/completely black legs, the cuticle gradually shrivelling to obtain a raspberry-like texture (see Carner, 1976; Dick et al., 1992) as the resting spores beneath became discernible. This process left the cadavers smaller than mummified cadavers of the same
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life stage. They contained between 30 and 220 spores each. Resting spore-filled cadavers were commonly fixed to the leaf either by their mouthparts, in the case of larger individuals, or along the length of the body by rhizoids, in the case of the smaller individuals. Many cadavers were loose on the leaf but bore rhizoids, apparently having become detached. Some cadavers appeared to be secured incidentally by surrounding material, such as fungal mycelium, arthropod silk or other mite cadavers and exuviae. The rhizoids were black or clear and were unbranched, extending in all directions on the leaf surface to approximately 0.8 mm from the body. Microscopic observation revealed rhizoids running through the legs of some mites. On the five entire plants examined, nine resting sporefilled cadavers were found on leaves, three on the
growing tip and seven on the stem. However, brief examination of other stems showed that it would be wrong to conclude that much higher numbers of resting spores on leaves are accompanied by similarly higher numbers on stems. Entire cadavers were often found to have dried and were easily broken even with gentle handling. Many were found already to have broken up on the leaves, often leaving clumps of spores or even individual spores on the leaves. 3.2. Resting spore-filled mites on leaves—density estimates This study began four days after initial detection of N. floridana in the field, by which time epizootics in the two cassava fields monitored were already in progress,
Fig. 1. Monitoring of numbers on leaves of active stage mites (M. tanajoa, ‘‘CMG’’) and mites killed by the fungus N. floridana, either mummified or bearing resting spores. Piritiba, Bahia, Brazil, 1995. Note that the second peak on some median leaves (d) is due to apical leaves becoming median as the plant grows.
S.L. Elliot et al. / Journal of Invertebrate Pathology 81 (2002) 148–157
as evidenced by the presence of mummies (Fig. 1). In the two fields, mean estimated densities of M. tanajoa fell from between 20 and 90 individuals per leaf at the start of the study (29 June), to below 5 per leaf within three weeks (Fig. 1). Meanwhile, the initial dominance of mummies and near-absence of resting spore-filled cadavers reversed through the epizootic, with the numbers of the latter peaking in all cases on 10 July. In Field 1, densities of live and Neozygites-killed mites were much higher in the upper stratum than the middle. Although nearly four times as many resting spore-filled cadavers were found as live mummies in coincident peaks of abundance, these could have been formed before the mummies and persisted whereas mummies would not persist for long as they would sporulate. Thus, this does not imply that more mites developed resting spores than mummies. In Field 2, the epizootic was clearly at a more advanced stage at the start of monitoring: densities of live M. tanajoa were already falling as were mummy densi-
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ties, apparently from initially higher levels in the upper stratum. Resting-spore-filled cadavers appeared in the final stages of the epizootic, peaking at 10–14 per leaf. In the first few days of monitoring, live mummies were found at over 6 per leaf in both strata. These levels dropped off more rapidly in the middle stratum than the upper, perhaps reflecting the higher mite densities. The second peak in the density of resting spore-filled cadavers observed in the middle stratum (30 July) reflected apical leaves bearing spores from the first peak becoming median leaves with the growth of new leaves at the apex. From mid-August, nearly two months after the start of the epizootic, M. tanajoa numbers began to recover in the absence of the pathogen, principally in the upper stratum. In the majority of cases, the between-leaf variance in the number of resting spore-filled cadavers was greater than that for mummies (Table 2). Although quite consistent, this was only statistically significant in the apical leaves of Field 2.
Table 2 Comparison of values of variance: mean ratios of square roots of counts of mummies and resting spore-filled cadavers (RSFC, in bold type) from cassava leaves during an epizootic Field
Leaves
29 June*
2 July
10 July
20 July
1
Apical
Mummies RSFC P
— —
0.744 1.063 n.s.
0.632 0.937 n.s.
0.633 0.933 n.s.
Mummies RSFC P
—
—
—
—
1.000 0.583 n.s.
0.500 1.000 n.s.
Apical
Mummies RSFC P
0.496 1.138 <0.05
0.195 0.667 <0.05
0.865 3.619 <0.01
0.800 0.857 n.s.
Median
Mummies RSFC P
—
1.292 1.209 n.s.
1.374 1.785 n.s.
—
Median
2
—
—
Values of P are from F tests for comparisons of variances (N ¼ 15 except *, where N ¼ 30). Table 3 Factors affecting resting spore formation in the field: minimal models obtained in multiple regression analyses Parameters available for model
Minimal model 1 Parameter estimates
Date Field Stratum Live mites Feeding injury Pathogen-killed mites Intercept Total d.f. F R2
Minimal model 2 Partial F
Parameter estimates
Partial F
+0.1933
14.40
+0.1260
7.537
+2.291
26.62
+0.3317
5.294
+1.043
40.87 +1.488 )2.872
9.239
)5.537 178 30.39 49.74
178 29.61 48.47
Numbers of mites on individual leaves bearing N. floridana resting spores was the explanatory variable, with numbers of mites on leaves with any N. floridana as the binomial denominator. Maximal models with all available parameters were simplified by backward and forward stepwise simplification. In model 2, feeding injury is replaced by number of pathogen-killed mites. See text for further explanation.
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A minimal model was obtained from the multiple regression analysis to explain just under 50% of the variance in numbers of resting spore-filled cadavers (Table 3). When transformed parameters were tested for inclusion in the model, none of them were improvements on their untransformed counterparts. The factors which emerged as explaining the variation were the date, position in the plant and amount of feeding injury, but not the field or mite density, live or dead. As feeding injury is an ordinal parameter and so not suited to this type of analysis, it was removed from the model and other parameters were tested for inclusion. This led to Model 2, with the number of pathogen-killed mites in place of injury, as the only parameter which could replace injury and still provide a significant contribution (tested by removal from the model) to explaining the variation. 3.3. Trapping of falling resting spore-filled mites Resting spore-filled cadavers were caught in the traps below plants throughout the three-week epizootic period, at a mean rate of 0.27 cadavers per trap-day (range 0.0–0.8). From mid-July, catches continued at a lower level until early August with one final cadaver at the end of that month. Over the entire study period, total catches were 5.2 and 9.8 cadavers per trap for Fields 1 and 2, respectively (Table 4). We compared these figures with those found from the leaves above. Taking the peak numbers of these cadavers found on the apical and median leaves as conservative estimates of the numbers formed per leaf, these figures were summed and multiplied by five as each trap had approximately five apical and five median leaves directly above it (Table 4). This gave conservative estimates of the numbers of resting spore-filled cadavers on the leaves directly above each trap and the discrepancy with those caught is clear. The
Table 4 Estimation of percentage of resting spore-filled cadavers (RSFC) produced on cassava leaves which reached traps on the ground Resting spore-filled cadavers
Field 1
Field 2
Total catch in traps Peak on apical leaves ( ¼ x) Peak on median leaves ( ¼ y) Estimate of number above each trap ¼ 5ðx þ yÞ Number caught as percentage of estimated number above
5.2 11.1 2.0 65
9.8 9.5 13.9 117
8.0%
8.4%
Data are numbers of such cadavers caught in traps versus densities found on leaves from the same field, assuming five apical and five median leaves above each trap.
implication is that less than 10% of the cadavers formed on the leaves reached the soil intact. Indeed, in the second half of July, when the weather was dry, filtration of the trap contents revealed a very large quantity of black spherical microscopic particles. Examination under the compound microscope was not possible but it is probable that these were resting spores released from disintegrating cadavers, suggesting that this may have been the fate of the majority of resting spores on the leaves. This is backed up by the examination of leaf litter collected in the subsequent dry season which yielded no evidence of Neozygites-killed mites. 3.4. Resting spore formation test Of the 779 mites challenged, 339 (43.5%) were killed by the fungus. Of these, normal mummies were produced from 257 (75.8%) mites which died on average 5.8 days post-inoculation. Resting spores were produced in 82 (24.2%) mites, these dying on average 7.2 days postinoculation. The routine production of mummies in parallel with this yielded no resting spores. Indeed, this
Table 5 Results of bioassays in the N. floridana mummy viability experiment, begun 10 September 1996 Treatment
T T T T T T T T T
¼ 25 °C, ¼ 25 °C, ¼ 25 °C, ¼ 30 °C, ¼ 30 °C, ¼ 30 °C, ¼ 35 °C, ¼ 35 °C, ¼ 35 °C,
Number of mummies which sporulated (N ¼ 6) (Mean number conidia per sporulated mummy)
SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg
Bioassay after 37 days—17 October, lasting 48 h
Bioassay after 62 days—11 November, lasting 72 h
Bioassay after 73 days—22 November, lasting 72 h
0 1 2 2 2 1 1 1 1
0 0 0 0 2 (1) 0 0 4 (2) 0
0 0 0 0 0 0 0 0 0
(1) (1.5) (1) (1) (4) (2) (1) (2)
Field—Soil Field—Stem
0 0
0 0
0 0
Control (T ¼ 8 °C, SD ¼ 7 mm Hg)
4 (27.5)
4 (292.5)
3 (131.7)
Mummies were collected from the treatments after 37, 62, and 73 days (17 October, 11 November, and 22 November, respectively) and tested for their ability to sporulate and produce conidia over 48 or 72 h in dark, saturated conditions.
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procedure has never yielded more than very occasional resting spore production (<1 mite in a thousand). No rhizoid formation was found in the laboratory, which was also the case for Neozygites sp. from the tetranychid Oligonychus pratensis (Banks) (Dick et al., 1992). 3.5. Tests of mummy viability Although viability was retained by most of the control mummies over the 73 days of the experiment, it was greatly reduced when they were stored in the field or in hot and dry conditions representative of field conditions (Table 5). In the field, all viability was lost after 37 days. In all laboratory treatments, both the number of mummies sporulating and the number of conidia which these mummies produced were reduced after 37 days and were zero by the end of the experiment. After 62 days, only mummies from two of the intermediate saturation deficit treatments (SD ¼ 13 mm Hg) retained any viability, but this was not in evidence 11 days later. The mummies were observed throughout to have dried up rather than to have sporulated, most notably so with those left in the field. In the field, the mean soil humidity during this period was 0.85% (range 0–3.57%) versus 6.88% (range 0.84–18.75%) in the previous wet season (from 14 April to 24 July).
4. Discussion The aim of this paper is to give insight into the means by which N. floridana from M. tanajoa survives during dry seasons, focussing on a semi-arid region of northeastern Brazil. 4.1. Which mechanism does N. floridana use to survive the dry season? Resting spores were found both in this study and in the previous year (Delalibera, 1996). While their role in dry season survival has not been fully demonstrated, it is probable that they form the principal means of survival as they are especially adapted to this function, and there is no indication of alternative means being viable. Meanwhile, the low viability of mummies after storage under hot and dry conditions indicates that they do not represent a reliable means to bridge long gaps between epizootics. There was some viability after 62 days in the hot and dry conditions in the laboratory, but very few conidia were then produced, and periods between epizootics can be considerably longer than this (Elliot et al., 2000). In the field there was no viability. Furthermore, climatic conditions during a dry season may be sufficiently cool and humid to stimulate sporulation of mummies (GJM unpubl. data) but not for sufficiently long to allow an epizootic. Finally, observations suggest that it may take
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weeks of suitable climatic conditions for N. floridana to reach densities that can be detected in the field (Elliot et al. in prep.). These last two points would lead us to expect a dormancy mechanism in surviving N. floridana structures. Attempts to stimulate germination of resting spores failed (three spores of many hundreds were observed to germinate) even when spores were left for up to 13 months in the field (SLE pers. obs.). This suggests that there is a dormancy mechanism for these spores. 4.2. Why are resting spores not found more often? If resting spores are important in dry season survival then it seems odd that they should be found so infrequently in the field. While they may not have been formed in large numbers in most instances, the possibility that they were simply not detected cannot be ruled out. It is very hard to distinguish mites filled with resting spores from other dead mites or other material on leaves. Work directed at counts of live mites and mummies can easily lead the worker to overlook structures for which he or she does not have a Ôsearch image.Õ The technicians helping in this study stated that they would not have distinguished the cadavers bearing spores had they not been pointed out. Whether some apparent absences of resting spores from epizootics can be explained by these difficulties is a moot point. Infected mites die after the production of these spores, so they should be detectable on mounts of live mites (e.g., Delalibera, 1996). However, this is balanced by the possibilities that death follows resting spore formation very quickly, and that there is a bias in choosing live mites to mount and examine for signs of disease away from the visually different spore-filled mites. In live mounts of mites made from fields where resting sporefilled cadavers were found in abundance, none were found in live mounts. Other studies of N. floridana have found resting spores to be rare or absent (Carner and Canerday, 1968; Humber et al., 1981; Kenneth et al., 1972; Ramaseshiah, 1971; Weiser and Muma, 1966). It may be in some of these cases that the structures were simply not observed, so it is suggested that this possibility be considered. Perhaps field-collected mites could be incubated to determine if resting spores will form, as has been done with other entomophthoraleans in insects (e.g., Thomsen and Eilenberg, 2000). In the year following this study, almost no resting spores were found even though the investigators were the same (Elliot et al., 2002). This, and a similar observation from Africa (Hountondji et al., 2002a) supports the idea that resting spores appear sporadically. Why this should be is a mystery. One possibility is that the environment in the study area is changing over the years (from local verbal reports in the absence of weather records), and becoming drier. Epizootics may be becoming rarer, and the conditions for producing resting spores may be
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harder to meet. Alternatively, this could be an essential aspect of the pathogenÕs ecology: a similar pattern of infrequent resting spore formation during entomophthoralean (principally N. fresenii) epizootics was observed in Aphis fabae Scopoli in broad bean in France (Rabasse and Dedryver, 1982). If resting spores represent the principal or only means of dry season survival, the production of negligible amounts of these structures in an epizootic can be regarded as a failure on the part of the pathogen to transmit itself to future host populations. 4.3. What stimulates resting spore formation? The appearance of resting spores in the later stages of the epizootic fits a pattern common in the Entomophthorales (e.g., Klubertanz et al., 1991). This switch to the production of resting spores may be adaptive at the end of an epizootic, with a reduced population of potential hosts, a climate which is changing or soon to change, or both (Hajek and Shimazu, 1996; Milner and Lutton, 1983; Mietkiewski et al., 1993; Newman and Carner, 1975). The high between-leaf variation in the numbers of mites with resting spores suggests that variation at the level of the populations on a leaf may determine the formation of these structures. More mites produced resting spores (relative to conidia) later in the epizootic, on the lower stratum, on leaves more injured by mite feeding and on leaves with higher concentrations of the fungus. Whether the high levels of spores found in nymphs merely reflects the age structure of the population following the first wave of mortality, or has physiological or even behavioral reasons, merits further investigation, as would potential differences between strains of N. floridana and different mite hosts. A particular feature to note is that the temperature during this study fluctuated between 15 and 25 °C versus 20 and 25 °C in the following year (SLE pers. obs.). The production of resting spores in the laboratory was achieved by simulating field conditions observed when these structures were found. A quarter of the mites which died from mycosis bore resting spores, which demonstrates that the prospects for studies or even medium-scale production in the laboratory are good. The next step would clearly be to look at the range of potential contributing factors and their effect on resting spore formation, as has been done successfully in other entomophthoralean pathogens, notably Entomophaga maimaiga (e.g., Hajek and Humber, 1997; Kogan and Hajek, 2000). 4.4. Fate of resting spores Put together, the observations made in this study favor the notion that the majority of resting spores fall free to the soil upon the break-up of cadavers, remaining dormant until a subsequent epizootic. Although some ca-
davers were found on plant stems where they might be ideally positioned to infect future hosts, there were very few of these. Many cadavers were fixed to leaves by rhizoids or by their mouthparts in the case of larger mites. Because the production of rhizoids requires resources, it is to be supposed that it is somehow adaptive. Hosts dying with their mouthparts fixed in the leaves may be incidental but may equally arise through pathogenmediated behavior or even an undetected fungal holdfast structure. Mummies are also commonly found fixed in this fashion. Potential benefits may arise from this if there is a requirement for the initial maturation of spores on the leaf rather than in the soil or if dispersal is improved. Cadavers did not persist on leaves until dry season leaf-fall, and no spore-filled cadavers were found on approximately 10 kg of dry leaves collected from fields in which resting spores had been observed. 4.5. Implications for biological control Since resting spores seem to be the principal means of survival of N. floridana between rainy seasons, their formation following introductions of the pathogen to new areas may be a critical feature in its successful establishment in a classical biological control programme. Indeed, given that they are probably able to persist in hot dry conditions and germinate in the wet season, they seem the ideal means of application of the fungus in new areas: they could be applied at any time of year, to sporulate and cause epizootics once rains come. Also, a soil bank of resting spores may be formed, in the event of none being formed from epizootics in one year.
Acknowledgments We thank Valter Silva and Adailson de Luna for help with the field work and the people of Sumare, Piritiba, Bahia, for permission to work in their fields. We also thank two anonymous reviewers for helpful comments on the manuscript. The work was funded by the IITA/ EMBRAPA Agreement for the Biological Control of Cassava Pests and SLE was partly supported by the Netherlands Foundation for the Advancement of Tropical Research (WOTRO). The EMBRAPA Centro rido de Pesquisa Agropecuaria do Tr opico Semi-A (CPATSA), Petrolina, Pernambuco, Brazil, provided logistical support
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S.L. Elliot et al. / Journal of Invertebrate Pathology 81 (2002) 148–157 Briggs, C.J., Godfray, H.C.J., 1996. The dynamics of insect–pathogen interactions in seasonal environments. Theoretical Population Biology 50, 149–177. Carner, G.R., 1976. A description of the life cycle of Entomophthora sp. in the two spotted spider mite. Journal of Invertebrate Pathology 28, 245–254. Carner, G.R., Canerday, T.D., 1968. Field and laboratory investigations with Entomophthora fresenii, a pathogen of Tetranychus spp. Journal of Economic Entomology 61, 956–959. Carson, F.T., 1931. Control of relative humidity in a small inclosed space. Paper Trade Journal 29, 71–74. Crawley, M.J., 1993. GLIM for Ecologists. Blackwell Science, Oxford. de Moraes, G.J., de Alencar, J.A., Lima, J.L.S.D., Yaninek, J.S., Delalibera Jr., I., 1993. Alternative plant habitats for common phytoseiid predators of the cassava green mite (Acari: Phytoseiidae, Tetranychidae) in northeast Brazil. Experimental and Applied Acarology 17, 77–90. de Moraes, G.J., Delalibera Jr., I., 1992. Specificity of a strain of Neozygites sp., (Zygomycetes: Entomophthorales) to Mononychellus tanajoa (Acari: Tetranichidae). Experimental and Applied Acarology 14, 89–94. Delalibera Jr., I., 1996. Producß~ao, Distribuicß~ao Geografica e Epizootiologia de Neozygites sp., Pat ogeno de Mononychellus tanajoa. MasterÕs Thesis, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’—ESALQ-USP, Piracicaba, Brazil. Delalibera Jr., I., de Moraes, G.J., Sosa-Gomez, D.R., 1999. Epizootias de Neozygites floridana (Zygomycetes, Entomophthorales) e din^ amica populacional de acaros fitoseıdeos predadores de Mononychellus tanajoa (Acari, Phytoseiidae e Tetranychidae) na Bahia. Revista Brasileira de Entomologia, S~ao Paulo 43, 287–291. Delalibera Jr., I., Sosa-Gomez, D.R., de Moraes, G.J., de Alencar, J.A., Farias-Ara ujo, W., 1992. Infection of Mononychellus tanajoa (Acari: Tetranychidae) by the fungus Neozygites sp. in northeastern Brazil. Florida Entomologist 75, 145–147. Dick, G.L., Buschmann, L.L., Ramoska, W.A., 1992. Description of a species of Neozygites infecting Oligonychus pratensis in the western Great Plains of the United States. Mycologia 84, 729–738. Elliot, S.L., de Moraes, G.J., Delalibera Jr., I., da Silva, C.A.D., Tamai, M.A., Mumford, J.D., 2000. Potential of the mite-pathogenic fungus Neozygites floridana (Entomophthorales: Neozygitaceae) for control of cassava green mite Mononychellus tanajoa (Acari: Tetranychidae). Bulletin of Entomological Research 90, 191–200. Elliot, S.L., de Moraes, G.J., Mumford, J.D., 2002. Importance of ambient saturation deficits in an epizootic of the fungus Neozygites floridana in the cassava green mite (Mononychellus tanajoa). Experimental and Applied Acarology, in press. Gandon, S., 1998. The curse of the pharaoh hypothesis. Proceedings of the Royal Society of London Series B—Biological Sciences 265, 1545–1552. Hajek, A.E., Humber, R.A., 1997. Formation and germination of Entomophaga maimaga azygospores. Canadian Journal of Botany 75, 1739–1747. Hajek, A.E., Shimazu, M., 1996. Types of spores produced by Entomophaga maimaiga infecting the gypsy moth, Lymantria dispar. Canadian Journal of Botany 74, 708–715. Hochberg, M.E., 1989. The potential role of pathogens in biological control. Nature 337, 262–265. Hountondji, F.C.C., Lomer, C.J., Hanna, R., Cherry, A.J., Dara, S.K., 2002a. Field evaluation of Brazilian isolates of Neozygites floridana (Entomophthorales: Neozygitaceae) for the microbial control of the cassava green mite in Benin, West Africa. Biocontrol Science and Technology 12, 361–370. Hountondji, F.C.C., Yaninek, J.S., de Moraes, G.J., Oduor, G.I., 2002b. Host specificity of the cassava green mite pathogen Neozygites floridana. Biocontrol 47, 61–66. Humber, R.A., de Moraes, G.J., Santos, J.M.d., 1981. Natural infection of Tetranychus evansi (Acari: Tetranychidae) by a
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Triplosporum sp., (Zygomycetes: Entomophthorales) in northeastern Brazil. Entomophaga 26, 421–422. Kogan, P.H., Hajek, A.E., 2000. In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga. Journal of Invertebrate Pathology 75, 193–201. Johnson, C.G., 1940. The maintenance of high atmospheric humidities for entomological work with glycerol–water mixtures. Annals of Applied Biology 27, 295–299. Kenneth, R., Wallis, G., Gerson, U., Plaut, H.N., 1972. Observations and experiments on Triplosporum floridanum (Entomophthorales) attacking spider mites in Israel. Journal of Invertebrate Pathology 19, 366–369. Klubertanz, T.H., Pedigo, L.P., Carlson, R.E., 1991. Influence of fungal epizootics on the biology and management of the two spotted spider mite (Acari: Tetranychidae) in soybean. Environmental Entomology 20, 731–735. Leite, L.G., Smith, L., Moraes, G.J., Roberts, D.W., 2000. In vitro production of hyphal bodies of the mite pathogenic fungus Neozygites floridana. Mycologia 92, 201–207. Mietkiewski, R., Balazy, S., van der Geest, L.P.S., 1993. Observations on a mycosis of spider mites (Acari: Tetranychidae) caused by Neozygites floridana in Poland. Journal of Invertebrate Pathology 61, 317–319. Milner, R.J., Lutton, G.G., 1983. Effect of temperature on Zoophthora radicans (Brefeld) Batko: an introduced microbial control agent of the spotted alfalfa aphid, Therioaphis trifolii (Monell) f. maculata. Journal of the Australian Entomological Society 22, 167–173. Newman, G.G., Carner, G.R., 1975. Factors affecting the spore form of Entomophthora gammae. Journal of Invertebrate Pathology 26, 29–34. Oduor, G.I., Yaninek, J.S., van der Geest, L.P.S., de Moraes, G.J., 1995. Survival of Neozygites cf. floridana (Zygomycetes: Entomophthorales) in mummified cassava green mites and the viability of its primary conidia. Experimental and Applied Acarology 19, 479– 488. Oduor, G.I., Yaninek, J.S., van der Geest, L.P.S., de Moraes, G.J., 1996. Germination and viability of capilliconidia of Neozygites floridana (Zygomycetes: Entomophthorales) under constant temperature, humidity, and light conditions. Journal of Invertebrate Pathology 67, 267–278. Rabasse, J.-M., Dedryver, C.A., 1982. Facteurs de limitations des populations dÕAphis fabae dans lÕouest de la France. IV. Nouvelles donnees sur le deroulement des epizooties a Entomophthoracees sur feverole de printemps. Entomophaga 27, 39–53. Ramaseshiah, G., 1971. Occurence of an Entomophthora on tetranychid mites in India. Journal of Invertebrate Pathology 18, 421–424. Thomas, M.B., Cbongboui, C., Lomer, C.J., 1996. Between-season survival of the grasshopper pathogen Metarhizium flavoviride in the Sahel. Biocontrol Science and Technology 6, 569–573. Thomsen, L., Eilenberg, J., 2000. Entomophthora muscae resting spore formation in vivo in the host Delia radicum. Journal of Invertebrate Pathology 76, 127–130. van der Geest, L.P.S., Elliot, S.L., Breeuwer, J.A.J., Beerling, E.A.M., 2000. Pathogens of mites. Experimental and Applied Acarology 24, 497–560. Weiser, J., Muma, M.H., 1966. Entomophthora floridana n. sp., Phycomycetes: Entomophthoraceae), a parasite of the Texas citrus mite, Eutetranychus banksi. Florida Entomologist 49, 155–159. Yaninek, J.S., de Moraes, G.J., Markham, R.H., 1989. Handbook on the Cassava Green Mite (Mononychellus tanajoa) in Africa. IITA, Ibadan, Nigeria. Yaninek, J.S., Saizonou, S., Onzo, A., Zannou, I., Gnanvossou, D., 1996. Seasonal and habitat variability in the fungal pathogens, Neozygites cf. floridana and Hirsutella thompsonii, associated with cassava mites in Benin, West Africa. Biocontrol Science and Technology 6, 23–33.
The role of resting spores in the survival of the mite-pathogenic fungus Neozygites floridana from Mononychellus tanajoa during dry periods in Brazil Sam L. Elliot,a,* John D. Mumford,b and Gilberto J. de Moraesc a
NERC Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK Environmental Science and Technology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK Departamento de Entomologia, Fitopatologia e Zoologia Agrıcola, Escola Superior de Agricultura ‘‘Luiz de Queiroz,’’ ESALQ/USP 13418-900, Piracicaba, SP, Brazil b
c
Received 15 January 2002; accepted 10 June 2002
Abstract Survival of pathogens during long periods of unfavorable conditions can be critical to their ecology and to their use in biological control. In northeastern Brazil, the mite pathogen Neozygites floridana must survive hot and dry conditions between wet seasons when it infects the cassava green mite Mononychellus tanajoa. We report on large numbers of mite cadavers bearing resting spores towards the end of epizootics in mid-1995. High within-leaf variability indicated that local factors may be important in determining resting spore formation. These spores remain in the host cadaver on a leaf until the cadaver breaks up, whereupon the spores fall freely to the soil, there to remain dormant. Laboratory simulation of field conditions led to ca. 25% of mycosed individuals bearing resting spores. Mummies (without resting spores) kept in hot and dry conditions showed little or no viability within 2 months, implying no role for survival over extended dry periods. It is proposed that resting spores form the principal means by which this pathogen survives the dry season in the study area. This has implications for its introduction to new areas in classical biological control. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Neozygites floridana; Cassava green mite; Mononychellus tanajoa; Resting spores; Entomophthorales; Tetranychidae; Overwintering; Biological control; Mummies
1. Introduction Although biologists working on temperate communities often ascribe little seasonality to tropical insect– pathogen systems (e.g., Briggs and Godfray, 1996), dry seasons near the equator can impose as much environmental variability on a pathogen population as do temperate winters. This is especially true of fungi but some are remarkably well adapted to surviving hot and dry conditions for long periods (e.g., Thomas et al., 1996). The survival of insect- or mite-pathogens for extended periods outside the host is thought to select for virulence and to maintain their effectiveness as biological control agents (Hochberg, 1989; Gandon, 1998). *
Corresponding author. Fax: +44-1344-873173. E-mail address: [email protected] (S.L. Elliot).
It is important to understand this aspect of pathogen ecology, and this is exemplified in the case of Neozygites floridana (Weiser & Muma) Remaudiere & Keller (Entomophthorales: Neozygitaceae) infecting the cassava green mite Mononychellus tanajoa (Bondar) (Acari: Tetranychidae). This fungus is of interest for biological control of this pest mite in both the Neotropics where they are endemic, and in Africa where the mite is exotic (Elliot et al., 2000; van der Geest et al., 2000; Yaninek et al., 1996). Epizootics of N. floridana in northeastern Brazil are sporadic, characterized by long periods of inactivity coinciding with dry seasons (Elliot et al., 2000). While N. floridana is capable of producing resting spores, these structures occur rarely and at low levels during epizootics (Delalibera et al., 1992, 1999; Elliot et al., 2000, 2002; Yaninek et al., 1996), raising the question of how this fungus survives dry seasons,
0022-2011/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 2 2 - 2 0 1 1 ( 0 2 ) 0 0 1 9 2 - 1
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especially in semi-arid environments such as northeastern Brazil. The production of thick-walled resting spores (sexual zygospores or asexual azygospores) by entomophthoralean fungi can be seen as a specific adaptation to survival in inhospitable conditions or in the absence of potential hosts, although additional benefits may accrue through the generation of novel genotypes in the sexual zygospores. However, other mechanisms of survival are potentially available to these fungi. Principal among these is the survival in mummified cadavers of the host (‘‘mummies’’). Note that we do not use the term ‘‘mummy’’ for resting spore-filled cadavers as we saw no evidence of mummification of the host cadaver. Other forms include the spores normally involved in the cycling of the pathogen, primary or secondary conidia, or as active infections in a different population of the host (Brandenburg and Kennedy, 1981) or in another host species. Mummies of N. floridana are actually sclerotia formed from mummified host cadavers and contain hyphal bodies of the fungus. These structures prevent desiccation of the fungus for short periods in the course of an epizootic. They have been implicated in the survival during harsh winters in temperate climes of N. floridana from the two spotted spider mite Tetranychus urticae Koch (Brandenburg and Kennedy, 1981). An isolate from M. tanajoa in Piritiba remained viable for up to 16 months at 5% relative humidity and 4 °C, but this was reduced to 6–7 months at the same humidity at 24 °C (Oduor et al., 1995). Although this latter study demonstrated the potential of the fungus to survive extended periods as mummies, it did not very closely reflect natural field conditions, where daily temperatures often exceed 35 °C and are much higher in the surface layers of the soil (SLE pers. obs.). The two spore forms produced in the asexual cycle, primary conidia and capilliconidia, are unsuitable for long-term persistence in dry conditions as they can only survive for short periods at high temperatures and low humidities (Oduor et al., 1995, 1996). Survival at a low prevalence in the host population seems unlikely as it was not detected during several years of monitoring of M. tanajoa populations in Piritiba (Elliot et al., 2000). Similarly, survival in alternative hosts is unlikely as the host mite is almost exclusively found on cassava (de Moraes et al., 1993) and this strain of the pathogen is very specific to M. tanajoa (de Moraes and Delalibera, 1992; Hountondji et al., 2002b; Leite et al., 2000). For these reasons, we suspect that N. floridana from M. tanajoa survives dry seasons as resting spores, despite their rarity. We report on finding mites filled with resting spores in mid-1995, describing their appearance and occurrence, and their immediate fate in the field. We also report on a preliminary and successful attempt to produce resting spores in vivo in the laboratory. To
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assess the potential of N. floridana to survive as mummies, we conducted a test of their long-term viability in the laboratory at a range of temperatures and saturation deficits intended to reflect field conditions, as well as on cassava stems and in soil in the field.
2. Materials and methods All fieldwork was carried out in growersÕ cassava fields in Piritiba, central Bahia, northeastern Brazil. All field-collected material was taken to the laboratory and examined within a few hours under a dissection microscope. 2.1. Resting spores on plants Following the initial detection of resting spore-filled M. tanajoa cadavers on cassava leaves in the study area on 25 June 1995, several thousand leaves were collected from more than 30 fields in the region, as well as five entire cassava stems (ca. 30 leaves per stem). Cadavers were examined. Mummies are easily recognizable as light brown. Any darker cadavers found on the leaf were removed and squashed on filter paper or microscope slides, or were mounted for subsequent microscopic investigation. Systematic observations were made in two cassava fields of local variety grouping Olho Roxo. Fields ‘‘1’’ and ‘‘2’’ were 50 m apart and were 4 and 18 months old and 1.5 and 2.0 m high, respectively. From 29 June to 19 September 1995, samples of 15–30 apical and median leaves were taken at irregular intervals from each field. The number of live M. tanajoa per leaf and the feeding injury from these mites was estimated. Total counts were made of the mummies and resting spore-filled cadavers per leaf. To obtain rough estimates of mite densities, these were estimated as scores for each leaf and then converted to previously calibrated means (estimates of 0, 1–25, 26–200, >200 mites per leaf receive scores of 0, 1, 2, 3 and conversion factors of 0, 10, 110, 350, respectively; Yaninek et al., 1989). Estimates of leaf injury were based on a visual estimate of percentage chlorosis where 0, no visible injury; 1 ¼ 1–25%; 2 ¼ 26–50%; 3 ¼ 51–75%; 4 ¼ 76–100%. These estimates were quick and were sufficient to characterize mite densities and feeding injury. The irregular timing of samples was due to lengthy counts (particularly of spore-filled cadavers) and limited time. To indicate how much local factors determined the formation of resting spores or mummies, variance:mean ratios were calculated from the counts per leaf (squareroot transformed) of resting spore-filled cadavers and mummies for each sample where both were found. Their variances were compared using F tests.
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A multiple regression analysis was conducted in GLIM version 4 (Royal Statistical Society, 1993; Crawley, 1993) to test whether some of the variation in the proportion of killed mites that formed resting spores could be explained by the measured parameters. Data for all individual leaves collected from 29 June to 20 July were put into one data set. The number of mites per leaf containing resting spores was taken as the explanatory variable with the total number of Neozygites-killed mites as the binomial denominator. A binomial error structure was incorporated in the modelling, with a logit link function. The independent variables were: sample date, field, stratum (apical or median), active mite densities, feeding injury and the numbers of Neozygites-killed mites. A maximal model was fitted to the data and then was simplified by a combination of backward and forward stepwise elimination of parameters as described by Elliot et al. (2000), but using v2 tests rather than F tests (Crawley, 1993). When parameters were removed from the model, their transformed equivalents were tested by inclusion where relevant. These were squares for mite densities and feeding injury, as these were scores, and square root transformations for dead mite numbers as these were counts. The data in the minimal model were highly over-dispersed (clumping of data points at the tails of frequency distributions). To account for this in the analysis, the scale parameter was adjusted according to Crawley (1993, p. 278) and the significance of terms was tested through F tests of the change in deviance upon their removal. 2.2. The immediate fate of resting spores To determine if resting spore-filled cadavers fell from leaves to the ground, traps were made from round plastic bowls of 0:18 m2 area and 20 cm height. Each had a row of three adjacent holes of 1 cm diameter bored in the base and covered with mite-proof gauze. Five of these traps were placed in each of the two fields, below cassava plant apices and slightly tilted to allow the drainage of rainwater. The traps were examined daily at the outset, then at intervals of up to eight days from 14 July (at this stage it was apparent that very little material would be lost from the traps as they were protected from wind and rain-splash by the cassava canopy). Material caught in the traps was removed with a hair brush and placed in individual vials containing 70% alcohol. The vials were taken to the field station and the contents filtered using filter paper. Examining the papers under the dissecting microscope, all objects resembling mites were collected and mounted in AmmanÕs Blue/HoyerÕs medium mixture for microscopic examination. To determine if resting spore-filled cadavers remained on fallen leaves, fallen and dried leaves were collected from cassava fields where resting spores had been found in abundance. Approximately 10 kg of leaf litter were
collected from three fields in the dry season following the epizootic. These were stored dry and examined for evidence of Neozygites-killed mites. 2.3. Resting spore formation in the laboratory To attempt to observe resting spore formation in the laboratory, the conditions under which they were found in the field were simulated as closely as possible. The conditions were: night temperature minima approximately 15 °C with relative humidity maxima approaching 100%; daytime temperature maxima approximately 25 °C with relative humidity minima 30–50%; photoperiod 11L:13D; overcast conditions; leaves with 30–50% chlorosis due to mite feeding damage; evidence of recently high mite and pathogen levels (exuviae, faeces, and sporulated cadavers), principally nymph cadavers bearing resting spores. Thus, 20 discs of 2 cm diameter were cut from field-collected cassava leaves bearing 30– 50% chlorosis from mite feeding. These were placed on water-soaked foam mats in petri dishes. Four laboratory-produced mummies were placed on each disc and left for 48 h in the dark at 20 °C, 90% RH. Thirty M. tanajoa nymphs were placed on each disc and these were then kept in a regime of 11 h light, 25 °C, 50% RH, and 13 h dark, 15 °C, 90% RH. The discs were observed daily and any dead mites were removed and mounted for examination under the microscope. This procedure was repeated once. Routine in vivo production of mummies was conducted concurrently using the same source of fungus and of mites and the same procedure to produce inoculum but using undamaged leaf material, young adult female mites, and a post-inoculation regime of continuous light, 25 °C, and approximately 70% RH. 2.4. Test of mummy viability Mummies were produced for this experiment using the routine in vivo procedure described above. This was timed so that all mummies used in the experiment were produced within five days of one another. Every day, new mummies were collected and either stored at 25 °C over pure glycerol (for the field experiment) or placed directly in the relevant storage vials (for the laboratory experiment; see below). To test the survival of mummies under a range of conditions in the laboratory, nine treatments combining three temperature and saturation deficit regimes were used, in addition to a control (8 °C, over pure glycerol) (Table 1). For each replicate, 10 ml of the appropriate solution of glycerol (Carson, 1931; Johnson, 1940) was placed in a black plastic film canister. Cotton wool was placed in the tube, leaving the base soaked in the glycerol and a dry cotton wool bed above, upon which to store the mummies. Fifteen mummies were placed in each vial, with 10 vials per
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Table 1 Temperature and saturation deficit treatments used in the laboratory experiment to test viability of N. floridana mummies over extended periods Temperature
Saturation deficit (mm Hg)
Relative humidity (%)
Glycerol concentration used (%)
25 °C
6 13 20
75 45 16
54 74 94
30 °C
6 13 20
81 59 37
41 63 80
35 °C
6 13 20
86 69 53
34 48 68
The corresponding relative humidities and the glycerol concentrations required to achieve these are shown.
treatment. Temperature and relative humidity were monitored in larger plastic boxes. For the field treatments, twenty mummies were placed in small white nylon bags, each within a larger, more porous, brown nylon bag. On 10 September, 1996, the bags were placed in a cassava field, either in the upper layer of soil, or at the base of a cassava plant. These were lightly covered with earth or tied to a plant stem, respectively. For weekly estimates of soil moisture, sub-samples of the top 1 cm of soil from the bases of five cassava plants were taken from three separate areas in the field. The samples were weighed, dried for 48 h at 105 °C, and re-weighed. The difference in weights was divided by the wet weight and multiplied by 100 to estimate percentage moisture. Means were taken of the three samples. After 37, 62, and 73 days, one unopened canister or bag was collected from each treatment (laboratory and field) and from the control. Six of the twenty mummies were taken at random from each, due to time constraints in assessing sporulation, and were placed on clean microscope slides in a dark water-saturated environment at 20 °C for 48 h. These were subsequently mounted with AmmanÕs Blue and examined under the microscope. The presence or absence of hyphal bodies, conidiophores and primary conidia was recorded for each mummy. A total count of primary conidia and capilliconidia was made on each slide.
3. Results 3.1. Resting spore-filled mites on plants—general observations Daily monitoring of the region of Piritiba led to the detection of epizootics of N. floridana on 25 June, and subsequently of resting spore-filled cadavers. These were very difficult to distinguish from other M. tanajoa which had also died and turned black. This was particularly true of mummies which had already sporulated as these
were rapidly colonised by saprophytic fungi. Many hours of handling material were needed to learn to distinguish between these cadavers with confidence and about a third of the resting spore-filled cadavers required microscopic examination for confirmation. Although full counts could take up to an hour for a leaf, many leaves could be examined very quickly. The maximum number of resting spore-filled cadavers found on a single leaf was 210. They were found in the later stages of epizootics in all the fields examined (>30) and were initially found in very low numbers of adult female mites. Subsequently, they were mostly observed at higher numbers predominantly in nymphs (there were incidentally very few adults left alive at this stage). None were found in larvae. This pattern appeared to reflect a wave of initially high mortality and population reduction among mature mites, followed by infection and mortality among the next generation as these matured from eggs or larvae. There was no visually apparent difference in the within-leaf distribution of mummies versus spore-filled cadavers, except that the latter were commonly found on the very edges of the leaf lobe borders where these were curled, and also on the leaf petiole where it joins the body of the leaf. Indeed, this served as a rapid indicator of the presence of resting spores in the field using a hand lens. Recently killed mites filled with resting spores were brown to grey, contrasting with the brown to yellow observed with recently killed and mummifying mites filled with hyphal bodies. A few live mites were found bearing clear immature resting spores. No dead mites were found with both resting spores and conidiophores or primary conidia—i.e., the sexual and asexual cycle seemed to be mutually exclusive within an individual host. Cadavers ultimately became pitch black, with colorless or partially/completely black legs, the cuticle gradually shrivelling to obtain a raspberry-like texture (see Carner, 1976; Dick et al., 1992) as the resting spores beneath became discernible. This process left the cadavers smaller than mummified cadavers of the same
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life stage. They contained between 30 and 220 spores each. Resting spore-filled cadavers were commonly fixed to the leaf either by their mouthparts, in the case of larger individuals, or along the length of the body by rhizoids, in the case of the smaller individuals. Many cadavers were loose on the leaf but bore rhizoids, apparently having become detached. Some cadavers appeared to be secured incidentally by surrounding material, such as fungal mycelium, arthropod silk or other mite cadavers and exuviae. The rhizoids were black or clear and were unbranched, extending in all directions on the leaf surface to approximately 0.8 mm from the body. Microscopic observation revealed rhizoids running through the legs of some mites. On the five entire plants examined, nine resting sporefilled cadavers were found on leaves, three on the
growing tip and seven on the stem. However, brief examination of other stems showed that it would be wrong to conclude that much higher numbers of resting spores on leaves are accompanied by similarly higher numbers on stems. Entire cadavers were often found to have dried and were easily broken even with gentle handling. Many were found already to have broken up on the leaves, often leaving clumps of spores or even individual spores on the leaves. 3.2. Resting spore-filled mites on leaves—density estimates This study began four days after initial detection of N. floridana in the field, by which time epizootics in the two cassava fields monitored were already in progress,
Fig. 1. Monitoring of numbers on leaves of active stage mites (M. tanajoa, ‘‘CMG’’) and mites killed by the fungus N. floridana, either mummified or bearing resting spores. Piritiba, Bahia, Brazil, 1995. Note that the second peak on some median leaves (d) is due to apical leaves becoming median as the plant grows.
S.L. Elliot et al. / Journal of Invertebrate Pathology 81 (2002) 148–157
as evidenced by the presence of mummies (Fig. 1). In the two fields, mean estimated densities of M. tanajoa fell from between 20 and 90 individuals per leaf at the start of the study (29 June), to below 5 per leaf within three weeks (Fig. 1). Meanwhile, the initial dominance of mummies and near-absence of resting spore-filled cadavers reversed through the epizootic, with the numbers of the latter peaking in all cases on 10 July. In Field 1, densities of live and Neozygites-killed mites were much higher in the upper stratum than the middle. Although nearly four times as many resting spore-filled cadavers were found as live mummies in coincident peaks of abundance, these could have been formed before the mummies and persisted whereas mummies would not persist for long as they would sporulate. Thus, this does not imply that more mites developed resting spores than mummies. In Field 2, the epizootic was clearly at a more advanced stage at the start of monitoring: densities of live M. tanajoa were already falling as were mummy densi-
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ties, apparently from initially higher levels in the upper stratum. Resting-spore-filled cadavers appeared in the final stages of the epizootic, peaking at 10–14 per leaf. In the first few days of monitoring, live mummies were found at over 6 per leaf in both strata. These levels dropped off more rapidly in the middle stratum than the upper, perhaps reflecting the higher mite densities. The second peak in the density of resting spore-filled cadavers observed in the middle stratum (30 July) reflected apical leaves bearing spores from the first peak becoming median leaves with the growth of new leaves at the apex. From mid-August, nearly two months after the start of the epizootic, M. tanajoa numbers began to recover in the absence of the pathogen, principally in the upper stratum. In the majority of cases, the between-leaf variance in the number of resting spore-filled cadavers was greater than that for mummies (Table 2). Although quite consistent, this was only statistically significant in the apical leaves of Field 2.
Table 2 Comparison of values of variance: mean ratios of square roots of counts of mummies and resting spore-filled cadavers (RSFC, in bold type) from cassava leaves during an epizootic Field
Leaves
29 June*
2 July
10 July
20 July
1
Apical
Mummies RSFC P
— —
0.744 1.063 n.s.
0.632 0.937 n.s.
0.633 0.933 n.s.
Mummies RSFC P
—
—
—
—
1.000 0.583 n.s.
0.500 1.000 n.s.
Apical
Mummies RSFC P
0.496 1.138 <0.05
0.195 0.667 <0.05
0.865 3.619 <0.01
0.800 0.857 n.s.
Median
Mummies RSFC P
—
1.292 1.209 n.s.
1.374 1.785 n.s.
—
Median
2
—
—
Values of P are from F tests for comparisons of variances (N ¼ 15 except *, where N ¼ 30). Table 3 Factors affecting resting spore formation in the field: minimal models obtained in multiple regression analyses Parameters available for model
Minimal model 1 Parameter estimates
Date Field Stratum Live mites Feeding injury Pathogen-killed mites Intercept Total d.f. F R2
Minimal model 2 Partial F
Parameter estimates
Partial F
+0.1933
14.40
+0.1260
7.537
+2.291
26.62
+0.3317
5.294
+1.043
40.87 +1.488 )2.872
9.239
)5.537 178 30.39 49.74
178 29.61 48.47
Numbers of mites on individual leaves bearing N. floridana resting spores was the explanatory variable, with numbers of mites on leaves with any N. floridana as the binomial denominator. Maximal models with all available parameters were simplified by backward and forward stepwise simplification. In model 2, feeding injury is replaced by number of pathogen-killed mites. See text for further explanation.
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A minimal model was obtained from the multiple regression analysis to explain just under 50% of the variance in numbers of resting spore-filled cadavers (Table 3). When transformed parameters were tested for inclusion in the model, none of them were improvements on their untransformed counterparts. The factors which emerged as explaining the variation were the date, position in the plant and amount of feeding injury, but not the field or mite density, live or dead. As feeding injury is an ordinal parameter and so not suited to this type of analysis, it was removed from the model and other parameters were tested for inclusion. This led to Model 2, with the number of pathogen-killed mites in place of injury, as the only parameter which could replace injury and still provide a significant contribution (tested by removal from the model) to explaining the variation. 3.3. Trapping of falling resting spore-filled mites Resting spore-filled cadavers were caught in the traps below plants throughout the three-week epizootic period, at a mean rate of 0.27 cadavers per trap-day (range 0.0–0.8). From mid-July, catches continued at a lower level until early August with one final cadaver at the end of that month. Over the entire study period, total catches were 5.2 and 9.8 cadavers per trap for Fields 1 and 2, respectively (Table 4). We compared these figures with those found from the leaves above. Taking the peak numbers of these cadavers found on the apical and median leaves as conservative estimates of the numbers formed per leaf, these figures were summed and multiplied by five as each trap had approximately five apical and five median leaves directly above it (Table 4). This gave conservative estimates of the numbers of resting spore-filled cadavers on the leaves directly above each trap and the discrepancy with those caught is clear. The
Table 4 Estimation of percentage of resting spore-filled cadavers (RSFC) produced on cassava leaves which reached traps on the ground Resting spore-filled cadavers
Field 1
Field 2
Total catch in traps Peak on apical leaves ( ¼ x) Peak on median leaves ( ¼ y) Estimate of number above each trap ¼ 5ðx þ yÞ Number caught as percentage of estimated number above
5.2 11.1 2.0 65
9.8 9.5 13.9 117
8.0%
8.4%
Data are numbers of such cadavers caught in traps versus densities found on leaves from the same field, assuming five apical and five median leaves above each trap.
implication is that less than 10% of the cadavers formed on the leaves reached the soil intact. Indeed, in the second half of July, when the weather was dry, filtration of the trap contents revealed a very large quantity of black spherical microscopic particles. Examination under the compound microscope was not possible but it is probable that these were resting spores released from disintegrating cadavers, suggesting that this may have been the fate of the majority of resting spores on the leaves. This is backed up by the examination of leaf litter collected in the subsequent dry season which yielded no evidence of Neozygites-killed mites. 3.4. Resting spore formation test Of the 779 mites challenged, 339 (43.5%) were killed by the fungus. Of these, normal mummies were produced from 257 (75.8%) mites which died on average 5.8 days post-inoculation. Resting spores were produced in 82 (24.2%) mites, these dying on average 7.2 days postinoculation. The routine production of mummies in parallel with this yielded no resting spores. Indeed, this
Table 5 Results of bioassays in the N. floridana mummy viability experiment, begun 10 September 1996 Treatment
T T T T T T T T T
¼ 25 °C, ¼ 25 °C, ¼ 25 °C, ¼ 30 °C, ¼ 30 °C, ¼ 30 °C, ¼ 35 °C, ¼ 35 °C, ¼ 35 °C,
Number of mummies which sporulated (N ¼ 6) (Mean number conidia per sporulated mummy)
SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg SD ¼ 6 mm Hg SD ¼ 13 mm Hg SD ¼ 20 mm Hg
Bioassay after 37 days—17 October, lasting 48 h
Bioassay after 62 days—11 November, lasting 72 h
Bioassay after 73 days—22 November, lasting 72 h
0 1 2 2 2 1 1 1 1
0 0 0 0 2 (1) 0 0 4 (2) 0
0 0 0 0 0 0 0 0 0
(1) (1.5) (1) (1) (4) (2) (1) (2)
Field—Soil Field—Stem
0 0
0 0
0 0
Control (T ¼ 8 °C, SD ¼ 7 mm Hg)
4 (27.5)
4 (292.5)
3 (131.7)
Mummies were collected from the treatments after 37, 62, and 73 days (17 October, 11 November, and 22 November, respectively) and tested for their ability to sporulate and produce conidia over 48 or 72 h in dark, saturated conditions.
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procedure has never yielded more than very occasional resting spore production (<1 mite in a thousand). No rhizoid formation was found in the laboratory, which was also the case for Neozygites sp. from the tetranychid Oligonychus pratensis (Banks) (Dick et al., 1992). 3.5. Tests of mummy viability Although viability was retained by most of the control mummies over the 73 days of the experiment, it was greatly reduced when they were stored in the field or in hot and dry conditions representative of field conditions (Table 5). In the field, all viability was lost after 37 days. In all laboratory treatments, both the number of mummies sporulating and the number of conidia which these mummies produced were reduced after 37 days and were zero by the end of the experiment. After 62 days, only mummies from two of the intermediate saturation deficit treatments (SD ¼ 13 mm Hg) retained any viability, but this was not in evidence 11 days later. The mummies were observed throughout to have dried up rather than to have sporulated, most notably so with those left in the field. In the field, the mean soil humidity during this period was 0.85% (range 0–3.57%) versus 6.88% (range 0.84–18.75%) in the previous wet season (from 14 April to 24 July).
4. Discussion The aim of this paper is to give insight into the means by which N. floridana from M. tanajoa survives during dry seasons, focussing on a semi-arid region of northeastern Brazil. 4.1. Which mechanism does N. floridana use to survive the dry season? Resting spores were found both in this study and in the previous year (Delalibera, 1996). While their role in dry season survival has not been fully demonstrated, it is probable that they form the principal means of survival as they are especially adapted to this function, and there is no indication of alternative means being viable. Meanwhile, the low viability of mummies after storage under hot and dry conditions indicates that they do not represent a reliable means to bridge long gaps between epizootics. There was some viability after 62 days in the hot and dry conditions in the laboratory, but very few conidia were then produced, and periods between epizootics can be considerably longer than this (Elliot et al., 2000). In the field there was no viability. Furthermore, climatic conditions during a dry season may be sufficiently cool and humid to stimulate sporulation of mummies (GJM unpubl. data) but not for sufficiently long to allow an epizootic. Finally, observations suggest that it may take
155
weeks of suitable climatic conditions for N. floridana to reach densities that can be detected in the field (Elliot et al. in prep.). These last two points would lead us to expect a dormancy mechanism in surviving N. floridana structures. Attempts to stimulate germination of resting spores failed (three spores of many hundreds were observed to germinate) even when spores were left for up to 13 months in the field (SLE pers. obs.). This suggests that there is a dormancy mechanism for these spores. 4.2. Why are resting spores not found more often? If resting spores are important in dry season survival then it seems odd that they should be found so infrequently in the field. While they may not have been formed in large numbers in most instances, the possibility that they were simply not detected cannot be ruled out. It is very hard to distinguish mites filled with resting spores from other dead mites or other material on leaves. Work directed at counts of live mites and mummies can easily lead the worker to overlook structures for which he or she does not have a Ôsearch image.Õ The technicians helping in this study stated that they would not have distinguished the cadavers bearing spores had they not been pointed out. Whether some apparent absences of resting spores from epizootics can be explained by these difficulties is a moot point. Infected mites die after the production of these spores, so they should be detectable on mounts of live mites (e.g., Delalibera, 1996). However, this is balanced by the possibilities that death follows resting spore formation very quickly, and that there is a bias in choosing live mites to mount and examine for signs of disease away from the visually different spore-filled mites. In live mounts of mites made from fields where resting sporefilled cadavers were found in abundance, none were found in live mounts. Other studies of N. floridana have found resting spores to be rare or absent (Carner and Canerday, 1968; Humber et al., 1981; Kenneth et al., 1972; Ramaseshiah, 1971; Weiser and Muma, 1966). It may be in some of these cases that the structures were simply not observed, so it is suggested that this possibility be considered. Perhaps field-collected mites could be incubated to determine if resting spores will form, as has been done with other entomophthoraleans in insects (e.g., Thomsen and Eilenberg, 2000). In the year following this study, almost no resting spores were found even though the investigators were the same (Elliot et al., 2002). This, and a similar observation from Africa (Hountondji et al., 2002a) supports the idea that resting spores appear sporadically. Why this should be is a mystery. One possibility is that the environment in the study area is changing over the years (from local verbal reports in the absence of weather records), and becoming drier. Epizootics may be becoming rarer, and the conditions for producing resting spores may be
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harder to meet. Alternatively, this could be an essential aspect of the pathogenÕs ecology: a similar pattern of infrequent resting spore formation during entomophthoralean (principally N. fresenii) epizootics was observed in Aphis fabae Scopoli in broad bean in France (Rabasse and Dedryver, 1982). If resting spores represent the principal or only means of dry season survival, the production of negligible amounts of these structures in an epizootic can be regarded as a failure on the part of the pathogen to transmit itself to future host populations. 4.3. What stimulates resting spore formation? The appearance of resting spores in the later stages of the epizootic fits a pattern common in the Entomophthorales (e.g., Klubertanz et al., 1991). This switch to the production of resting spores may be adaptive at the end of an epizootic, with a reduced population of potential hosts, a climate which is changing or soon to change, or both (Hajek and Shimazu, 1996; Milner and Lutton, 1983; Mietkiewski et al., 1993; Newman and Carner, 1975). The high between-leaf variation in the numbers of mites with resting spores suggests that variation at the level of the populations on a leaf may determine the formation of these structures. More mites produced resting spores (relative to conidia) later in the epizootic, on the lower stratum, on leaves more injured by mite feeding and on leaves with higher concentrations of the fungus. Whether the high levels of spores found in nymphs merely reflects the age structure of the population following the first wave of mortality, or has physiological or even behavioral reasons, merits further investigation, as would potential differences between strains of N. floridana and different mite hosts. A particular feature to note is that the temperature during this study fluctuated between 15 and 25 °C versus 20 and 25 °C in the following year (SLE pers. obs.). The production of resting spores in the laboratory was achieved by simulating field conditions observed when these structures were found. A quarter of the mites which died from mycosis bore resting spores, which demonstrates that the prospects for studies or even medium-scale production in the laboratory are good. The next step would clearly be to look at the range of potential contributing factors and their effect on resting spore formation, as has been done successfully in other entomophthoralean pathogens, notably Entomophaga maimaiga (e.g., Hajek and Humber, 1997; Kogan and Hajek, 2000). 4.4. Fate of resting spores Put together, the observations made in this study favor the notion that the majority of resting spores fall free to the soil upon the break-up of cadavers, remaining dormant until a subsequent epizootic. Although some ca-
davers were found on plant stems where they might be ideally positioned to infect future hosts, there were very few of these. Many cadavers were fixed to leaves by rhizoids or by their mouthparts in the case of larger mites. Because the production of rhizoids requires resources, it is to be supposed that it is somehow adaptive. Hosts dying with their mouthparts fixed in the leaves may be incidental but may equally arise through pathogenmediated behavior or even an undetected fungal holdfast structure. Mummies are also commonly found fixed in this fashion. Potential benefits may arise from this if there is a requirement for the initial maturation of spores on the leaf rather than in the soil or if dispersal is improved. Cadavers did not persist on leaves until dry season leaf-fall, and no spore-filled cadavers were found on approximately 10 kg of dry leaves collected from fields in which resting spores had been observed. 4.5. Implications for biological control Since resting spores seem to be the principal means of survival of N. floridana between rainy seasons, their formation following introductions of the pathogen to new areas may be a critical feature in its successful establishment in a classical biological control programme. Indeed, given that they are probably able to persist in hot dry conditions and germinate in the wet season, they seem the ideal means of application of the fungus in new areas: they could be applied at any time of year, to sporulate and cause epizootics once rains come. Also, a soil bank of resting spores may be formed, in the event of none being formed from epizootics in one year.
Acknowledgments We thank Valter Silva and Adailson de Luna for help with the field work and the people of Sumare, Piritiba, Bahia, for permission to work in their fields. We also thank two anonymous reviewers for helpful comments on the manuscript. The work was funded by the IITA/ EMBRAPA Agreement for the Biological Control of Cassava Pests and SLE was partly supported by the Netherlands Foundation for the Advancement of Tropical Research (WOTRO). The EMBRAPA Centro rido de Pesquisa Agropecuaria do Tr opico Semi-A (CPATSA), Petrolina, Pernambuco, Brazil, provided logistical support
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