Microb Ecol DOI 10.1007/s00248-008-9477-5 ORIGINAL ARTICLE Fungal Phyllosphere Communities are Altered by Indirect Interactions Among Trophic Levels Jose L. Perez & J. Victor French & Kenneth R. Summy & Anita Davelos Baines & Christopher R. Little Received: 25 January 2008 / Accepted: 10 November 2008 # Springer Science + Business Media, LLC 2008 Abstract Trophic interactions involving predators, herbivores, and plants have been described in terrestrial systems. However, there is almost no information on the effect of trophic interactions on microbial phyllosphere community abundance, diversity, or structure. In this study, the interaction between a parasitoid, an insect herbivore, and the fungal phyllosphere community is examined. Parasitoid wasps have an indirect negative impact on fungal community diversity. On the citrus phyllosphere, the exotic wasp species, Amitus hesperidum and Encarsia opulenta, may parasitize the citrus blackfly (Aleurocanthus woglumi). If parasitism levels are low, the blackfly may produce significant amounts of honeydew secretions on the surface of the leaf. Honeydew deposition provides a carbon-rich substrate for the development of fungal growth persisting as sooty mold on the leaves. Leaves from sooty mold-infested grapefruit (Citrus paradisi) trees were collected from multiple orchards in south Texas. The effect of different levels of exotic parasite activity, citrus blackfly, and sooty mold infestation on phyllosphere mycobiota community structure and diversity was examined. Our results suggest the presence of the parasitoid may lead to a top–down trophic cascade affecting phyllosphere fungal community diversity and structure. Additionally, persistent sooty mold deposits J. V. French Texas A&M University—Kingsville Citrus Center, Weslaco, TX 78596, USA J. L. Perez : K. R. Summy : A. D. Baines Department of Biology and Center for Subtropical Studies, The University of Texas—Pan American, Edinburg, TX 78541, USA C. R. Little (*) Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA e-mail: crlittle@ksu.edu that have classically been referred to as Capnodium citri (and related asexual morphological forms) actually comprise a myriad of fungal species including many saprophytes and potential fruit and foliar pathogens of citrus. Introduction The phyllosphere may be defined as that part of the leaf serving as the interface between the plant organ and the environment. Although the phyllosphere has been referred to as a relatively “hostile environment”, a number of macroand microorganisms successfully exploit this niche [38, 63]; thus, it serves as a microcosm for a complex series of multitrophic interactions. However, few studies have focused on the interactions between macrobiotic herbivores, the honeydew that some produce and the dynamics and diversity of phyllosphere microorganisms [43, 56, 57]. The role that other trophic levels may play in this interaction has not been explored. A good model system for exploring the complex dynamics of phyllosphere multitrophic interactions involves citrus, its indigenous phyllosphere fungi, the herbivorous citrus blackfly, Aleurocanthus woglumi Ashby (an important pest of citrus), and parasitic wasps of the herbivore. The mycobiota (fungal epiphytes) of citrus leaves normally include a number of moniliaceous (nonmelanized) and dematiaceous (melanized) hyphomycetes, coelomycetes that produce asexual fruiting structures such as pycnidia or acervuli, many ascomycetes and several yeast/dimorphic forms [1, 5, 22, 23, 41, 47, 61, 63]. Sooty mold is the direct result of saprophytic fungal growth upon the copious honeydew excretions that result from the feeding activities of homopteran insects [8, 10, 39, 56]. These include such pests as whiteflies (Aleyrodidae; including the “blackfly”, mentioned above), aphids (Aphididae), mealybugs (Pseudococcidae), and soft scales (Coc- J. L. Perez et al. cidae) [19, 27, 34]. High levels of sooty mold on commercial citrus fruit can result in loss of fruit quality and economic losses for the grower [2]. Further, presence of sooty mold on leaves may interfere with photosynthesis and other physiological functions of the leaf resulting in reduced plant growth, fruit set, and yield [19, 60]. When populations of honeydew-excreting insects are under effective regulation by associated natural enemies, densities rarely increase to levels where honeydew becomes an economic problem. However, when the latter are disrupted (e.g., by insecticidal drift or overuse), densities of honeydew-excreting insects typically increase to levels where both honeydew and sooty mold deposits become damaging to the host plant [59]. In the case of A. woglumi (citrus blackfly, CBF), two exotic parasitic wasps, Amitus hesperidum Silvestri and Encarsia opulenta Silvestri are host-specific and may significantly decrease CBF populations [59]. We hypothesize that the mycofloral dynamics of the citrus phyllosphere are greatly altered when insects, such as the CBF, excrete honeydew upon leaves as a result of their feeding activities. This interaction is complicated by the potential for macrobiotic inhabitants of the phyllosphere to have specific parasites, which in some cases have been introduced as a mechanism of biocontrol. Indirect interactions among trophic levels in food webs have been described for a variety of terrestrial and aquatic ecosystems. For example, trophic cascades are indirect mutualisms between nonadjacent levels in a food chain whose effects may be either top–down (driven by a predator) or bottom–up (driven by primary producers) [44]. In terrestrial systems, the focus of many of these studies has been on the top–down effects of a predator on plant community diversity and structure (e.g., [7]). Few studies in terrestrial systems have investigated indirect effects among trophic levels within the phyllosphere. The purpose of this study was to compare fungal community structure and diversity on the citrus phyllosphere exhibiting various levels of sooty mold development caused by A. woglumi feeding activities and honeydew deposition. Overall, these data provide insight into direct and indirect effects among trophic levels in this complex interaction. Also, we suggest that the effects of parasitic wasps on fungal community structure and diversity may represent a trophic cascade in this system. Methods Sampling Locations and Visual Estimations of Orchards Grapefruit (Citrus paradisi) leaf samples were collected from 97 sample sites (1 “site” = individual tree within an orchard) throughout the grapefruit-producing areas in the Lower Rio Grande Valley (LRGV) of Texas during March of 2004. Leaf samples were taken from orchards that were infested with citrus blackfly (CBF) and heavily covered with sooty mold, although levels of sooty mold varied on individual leaves. These orchards were located within an ~240 km2 (150 mi2) area and were operated by Rio Queen Citrus (Mission, Texas; 57 sample sites), Smith Grove Care (Mission, Texas; 19 sample sites), Healds Valley Farms (Edinburg, Texas; eight sample sites), and several independent growers (Mission and Donna, Texas; 13 sample sites). A total of seven different orchards were sampled. Each sampling site was a tree, and each sample consisted of ten to 15 citrus leaves and was selected from trees in areas of the orchards that appeared to have relatively high CBF infestations. The point of this study was not to compare variability within or between orchards, but to characterize the variability of the interaction through broad geographical sampling (as indicated above). The samples were rated visually for three categories: (1) level of CBF infestation [“CBF” (total CBF)], (2) visual sooty mold rating (“VSMR”), and (3) level of CBF parasitism [“PEH” (percent pupae with parasite exit holes)]. The presence or absence of adult parasites was also recorded. The visual estimations of the samples were based on a form distributed to grapefruit growers in the Lower Rio Grande Valley by the Texas Citrus Mutual producers organization. Isolation of Culturable Fungi from Grapefruit Phyllospheres Five leaf sections were obtained arbitrarily from leaves collected from each of the 97 sampling sites; one leaf section per leaf for a total of five leaf sections per site. After collection, leaf material was stored at 4°C for no more than 7–10 days prior to suspension of epiphyte propagules. Each leaf section measured 2 cm2 and was dissected from an individual leaf with a flame-sterilized scalpel. The five leaf sections were placed in 10 ml of a 0.5% sterilized gelatin solution and vortexed for 30 s at high speed in order to dislodge conidia from the leaf surface. Afterward, 100 μl of the vortexed solution were spread-plated onto potato dextrose agar medium containing ampicillin (20 μg/ml) and streptomycin (10 μg/ml), in order to prevent bacterial growth. Plates that were seeded with the phyllosphere solution were allowed to incubate at 25°C on a 16 h light:8 h dark cycle. After 3 days of incubation, the number of total fungal colonies was counted, and the morphology of each was recorded. Pure cultures of each unknown were prepared using standard microbiological techniques. Culture plates were stored at 4°C while not in use. Phyllosphere Fungal Communities Fungal Identification After fungi were isolated, they were subcultured on potato dextrose agar (PDA). Fusarium spp. were grown on Nash– Snyder medium [NS: 15 g peptone, 20 g agar, 1 g KH2PO4, 0.5 g MgSO4, 1 g PCNB (pentacholoronitrobenzene) per liter of H2O]. Single spore isolates of Fusarium were generated on PDA. For identification, carnation leaf agar (CLA) was used to promote the production of microconidia and macroconidia (if present) [9, 37]. Penicillium spp., Aspergillus sp., and Paecilomyces sp. were grown on a set of differential media, including Czapek’s–Dox agar (CA), Czapek’s–Dox yeast extract agar (CYA), and malt extract agar (MEA) [48]. The remaining groups including the Capnodiales and cleistothecial/pseudothecial ascomycetes (CPAs) were grown on PDA and Sabouraud’s dextrose agar (SDA) amended with antibiotics (see above). All fungi that were identified were morphologically characterized using light microscopy at ×50–400. Yeasts were grown on yeast peptone dextrose agar (YPD). Yeasts were broadly grouped based on color into those that produced dark colonies (black yeasts), pink colonies (red yeasts), and cream colonies (white yeasts). It was not the aim of this study to narrowly classify the yeast from citrus leaves, but rather to look at the broad groups as outlined. Contingency and Correlation Analyses Contingency analysis was performed to examine the association between VSMR, CBF, PEH, and adult parasites (PROC FREQ; SAS Institute Inc., [53]). Comparisons among total samples (N=97) were conducted in all possible combinations for total CBF, VSMR, and PEH using Spearman’s rank correlation [55]. Estimations of Diversity Shannon’s and Simpson’s diversity indices were calculated from pooled leaf samples for “none” (0), “light” (1), “medium” (2), and “heavy” (3), sooty mold infestations. The Simpson’s inverse diversity index (dominance index) is calculated based on the formula: 1=DS , where DS ¼ 1
P ½ ni ðni
1Þ=NðN
1Þ, in which ni is the number of isolates in a group, and N is the total number of isolates. The Simpson’s index gives strong weighting to dominant members of the community of interest [30]. The Shannon’s P diversity
. index is based on the formula H ¼
xi =xo l nxi xo where xi is the number of isolates in a group  and xo is the total number of isolates. The Shannon index provides a moderate weighting to rare and intermediate species in comparison to dominant species. In this way, the Shannon index is more sensitive to changes in abundance of rare groups compared to other diversity indices [30]. Diversity indices and standard deviations were calculated using EstimateS [14]. Differences in mean CFUs among VMSR, CBF, and PEH categories were examined with analysis of variance (PROC GLM; SAS Institute Inc., [54]). Difference in mean CFUs with presence or absence of adults was examined with a t test (PROC TTEST; SAS Institute Inc., [53]). Results Leaf samples collected during 2004 revealed the presence of active A. woglumi infestations in all 97 sampling sites, with CBF (citrus blackfly) levels ranging from “light” (48.5%) to “heavy” (25.8%; Table 1). Visual sooty mold ratings (VSMR) of individual leaves within these orchards were variable, ranging from “none” (8.2%) to “heavy” (55.7%). Based on a visual survey, populations of parasites (primarily E. opulenta) appeared to have been increasing in many orchards at the time the samples were collected; evidence of parasitism was not detected in 24.7% of sampling sites, and levels of parasitism (based on parasite exit holes) in the remainder ranged from “light” (50.5%) to “heavy” (17.5%). Relationships between sooty mold ratings and host–parasite densities and community composition and diversity of fungi on the citrus phyllosphere are summarized in the following sections. Visual Estimations of Citrus Orchards Grapefruit leaf samples were obtained from orchards and assigned categories for each of the following attributes: total citrus blackfly (CBF), visual sooty mold rating (VSMR), percent parasite exit holes (PEH; found in CBF pupae), and presence of adult parasites (either A. hesperidum or E. opulenta). The legend of Table 1 gives a brief description of each visual assessment category used in evaluating leaf samples. The samples obtained came from orchards that were thought (prior to sampling) to have heavy infestations of CBF. Sampling was performed in this directed way to ensure that all of the members of the interaction would be present. However, most of the leaf samples (48.5%) were rated as “light” (1) indicating that CBF was found in only isolated portions of orchards in a few trees (Table 1). The remaining samples were classified as “medium” (2) or “heavy” (3), indicating that about half of the total samples were from orchards showing immature and/or mature CBF on a widespread number of trees (Table 1). Although “light” (1) amounts of CBF were obtained from most of the samples, the level of sooty mold deposition was classified as “heavy” (3) in most leaf samples (55.7%; Table 1). This measure indicates orchard-wide infestations of sooty mold J. L. Perez et al. Table 1 Visual ratings and contingency analysisa of citrus blackfly (CBF), sooty mold (VSMR), percent parasite exit holes (PEH), and emerged adult parasites (Adult) from 97 C. paradisi sample sites Noneb Obsf Total CBF VSMR PEH Adulth 0 8 24 60 (0.0%)h (8.2%) (24.7%) (61.9%) Lightc Mediumd Heavye Expg Obs Exp Obs Exp Obs 10.7 10.7 10.7 48.5 47 17 49 37 37.6 37.6 37.6 48.5 25 (25.8%) 18 (18.6%) 7 (7.2%) – 16.7 16.7 16.7 25 (25.8%) 54 (55.7%) 17 (17.5%) – (48.5%) (17.5%) (50.5%) (38.1%) Exp 32 32 32 Contingency analysis was performed between VSMR, CBF, and PEH (χ2 =35.7, df=3, P<0.0001) (see “Results” section); “Adult” was not included in the analysis b None CBF, VSMR, PEH negligible or not present c Light immature CBF and sooty mold deposits highly localized on only a few trees, <10% of CBF pupae have PEH d Medium immature CBF and sooty mold deposits detectable in parts of orchards but absent in others, 25–50% of CBF pupae have PEH e Heavy high numbers of immature CBF evident on most leaves in orchard, sooty mold deposits heavy throughout orchard, >50% of CBF pupae have exit holes f Observed (“Obs”) and expected (“Exp”) counts ðExp ¼ ½column total  row total=overallÞ g Total leaf samples falling into visual rating category (percent of total leaf samples) (values are rounded to the nearest tenth of a percent) h Adult parasites were either present (1) or absent (0), (χ2 =5.66, df=1, P=0.017) a that were also heavy on a leaf-by-leaf basis. In general, less than 25% of CBF pupae had “medium” (2) or “heavy” (3) levels of parasitic activity (Table 1). Most leaf samples (51.5%) were rated as “light” (1) for percent parasite exit holes (Table 1). In addition, 38.1% of the samples showed the presence of adult parasites and 61.9% did not (Table 1; χ2 =5.66; df=1; P=0.017). Relationships Among Rating Categories There was a significant association between VSMR, CBF, and PEH (χ2 =35.7, df=3, P<0.0001; Table 1). Higher than expected CBF counts and lower than expected PEH counts were found at high VSMR levels (medium and heavy) (data not shown). A strong positive correlation was detected between numbers of CBF pupae on leaves and visual sooty mold ratings from the 97 leaf sample sites examined (Spearman’s Rs =+0.548; P<0.001; Table 2). CBF population density increases have a direct effect upon levels of honeydew on foliage that, in turn, directly affects levels of sooty mold (i.e., fungal population sizes). In addition, there was a positive correlation (Spearman’s Rs = 0.214; P=0.036) between VSMR ratings and the presence of parasite exit holes (Table 2). This association may be due to increased numbers of CBF, which will support greater parasite activity and adult parasites. However, this result does not signify that CBF populations are being regulated by the parasites. The direct effects of increased CBF populations on parasite populations show increased parasite activity (as measured by percent parasite exit holes). The presence of adult E. opulenta and parasite exit holes in CBF pupae on leaf samples (38.1% and 75.2%, respectively) demonstrates that parasite populations had dispersed into most of the CBF host populations at the time the samples were collected (Table 1). No significant correlation was detected between the numbers of the CBF hosts on leaves and parasite exit holes at this point in time (Spearman’s Rs =−0.006; P=0.953; Table 2). Community Structure of Culturable Phyllosphere Fungi A wide range of fungal groups predominated the isolations from leaves including anamorphic representatives from the Phylum Ascomycota (orders Capnodiales, Hypocreales, and Eurotiales). Also, a number of yeast and dimorphic forms, both ascomycetous and basidiomycetous, were isolated. Overall, the largest proportion of isolates (43.9%) was from the order Capnodiales (Table 3). The primary fungal species isolated and identified from this group was Capnodium citri (species complex; see “Discussion” section). Further, this group was found in the highest proportion in all VSMR categories, but the relative abundance was significantly higher only for VSMR category 2 (“medium”; Fig. 1). Members of the Hypocreales represented the second largest proportion (37.5%) of isolates from sooty molded grapefruit leaves (Table 3). The fungi isolated and identified from this group were Fusarium Table 2 Spearman rank correlation coefficients between visual ratings of citrus blackfly (CBF), sooty mold (VSMR), and percent parasite exit holes (PEH) from 97 C. paradisi leaf sample sites Total CBF VSMR PEH a Total CBF VSMR PEH 1.000 – – 0.5478 (P<0.001)a 1.000 – −0.0060 (P=0.953) 0.2136 (P=0.036)a 1.000 Significant correlation values (P<0.05) are followed by levels of probabilities (in parentheses) Phyllosphere Fungal Communities Table 3 Fungal groups obtained from sooty-molded C. paradisi phyllospheres in this study Visual sooty mold rating (VSMR) Fungal groups Capnodiales Capnodium spp. Hypocreales Fusarium spp. Eurotiales Penicillium spp. Paecilomyces spp. Aspergillus spp. NCDHsc CPAs Dimorphic Geotrichum spp. Aureobasidium spp. Yeastsd Y1 Y2 Y3 Y4 Unidentified formse SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 Total Nonea Lighta Mediuma Higha 15 (32.6%)b 1,108 (44.7%) 1,444 (48.5%) 6,410 (42.9%) 328 (13.2%) 1,117 (37.5%) 6,211 (41.6%) 9 (19.6%) 0 0 0 0 0 (0.0%) (0.0%) (0.0%) (0.0%) (0.0%) 0 3 16 0 1 15 0 3 1 0 2 0 0 0 0 0 0 46 (0.0%) (6.5%) (34.8%) (6.5%) 70 0 2 0 0 (2.8%) (0.0%) (< 0.1%) (0.0%) (0.0%) 0 (0.0%) 107 (4.3%) 717 (28.9%) 0 22 695 0 145 (5.9%) 0 0 0 0 130 3 2 10 0 2,477 263 7 0 4 0 (8.8%) (0.2%) (0.0%) (0.1%) (0.0%) 3 (0.1%) 3 (0.1%) 38 (1.3%) 0 4 27 7 96 (3.2%) 0 9 37 24 0 15 0 8 3 2,975 944 149 0 43 13 (6.3%) (1.0%) (0.0%) (0.3%) (< 0.1%) 101 (0.7%) 0 (0.0%) 108 (0.7%) 11 79 13 5 961 (6.4%) 48 125 157 18 0 0 445 125 43 14,940 a Total leaf samples for each VSMR group: none (8), light (17), medium (18), and high (54) Total CFUs (% CFUs (proportional abundance) in that category for each respective VSMR column) c NCDH non-capnodiaceous dematiaceous hyphomycetes, CPA cleistothecial or pseudothecial ascomycetes d Y1–Y4 unidentified, but morphologically distinct yeasts e SP1–SP9 unidentified, but morphologically distinct filamentous fungi b spp. including Fusarium moniliforme sensu lato [56], Fusarium solani, and Fusarium oxysporum. The relative abundance of this group increased significantly with VSMR ratings of “medium” or “heavy” (Fig. 1). Also present were members of the Eurotiales (7.0%) (Table 3). The relative abundance of Penicillium, Paecilomyces, and Aspergillus increased in samples that had higher VSMR ratings (Fig. 1). The primary species isolated of the penicillia included Penicillium digitatum, Penicillium italicum, Penicillium ulaiense, and Penicillium expansum. A Paecilomyces sp. was isolated frequently from leaves with VSMR categorizations of “medium” and “heavy,” although this isolate was not identified to species. The only species of Aspergillus isolated was A. niger. A number of yeasts were isolated from citrus leaves. Yeasts represented 4.3% of the total isolated fungi (in terms of colony-forming units; Table 3). These yeasts include both ascomycetous and basidiomycetous forms. Initial investigations suggest that the two most prominent yeast genera are Rhodotorula spp. (a red yeast) and at least one unidentified cryptococcoid form. Some dimorphic fungi categorized here also include Aureobasidium pullulans and an unidentified sporotrichoid form. Interestingly, the relative population of yeasts appears to decrease in leaf samples with increasing visual sooty mold ratings (Fig. 1). Species Richness and Diversity A total of 22 fungal subgroups were isolated from citrus leaves in this study. Species richness values (e.g., number of subgroups out of the total isolated) for VSMR, CBF, and PEH categories were compared (Table 4). As VSMR J. L. Perez et al. Figure 1 Proportional abundance of Capnodiales, Hypocreales, Eurotiales, and yeasts on the Citrus paradisi phyllosphere across four visual sooty mold rating (VSMR) categories [e.g., “none (0),” “light (1),” “medium (2),” and “heavy (3)”]. Values differ from their respective controls [VSMR category=“none (0)”] at P<0.05 (*), P< 0.01 (**), or P≤0.001 (***) according to the chi-square test increased, species richness increased (r=0.983, P=0.017), and as CBF increased, species richness remained relatively stable (r=0.240, P=0.846), except for “medium”, where there were 12 subgroups. As PEH increased, there was a decrease in species richness (r=−0.856, P=0.144). For the VSMR category of “none”, fungal communities were most diverse as determined by both Simpson’s and Shannon’s indices of diversity (Table 4). Lowest diversity was found at medium levels of sooty mold (VSMR). In contrast, highest fungal community diversity was observed for “medium” levels of parasite exit holes (Table 4). For CBF categories, the two measures of diversity gave inconsistent results with “light” having highest fungal community diversity for Simpson’s index and “heavy” having highest diversity for the Shannon’s index (Table 4). No significant differences in mean CFUs were found among CBF or PEH categories (F2,87 =0.35 and F3,87 =0.11, respectively). However, significant differences were found for VSMR categories (F3,87 =13.19) with “heavy” having significantly greater mean CFUs and “none” have significantly fewer. Mean CFUs were significantly higher in the presence of adult parasites (t=2.31, P<0.024). Discussion Recent outbreaks of citrus blackfly, A. woglumi, on citrus in the LRGV of south Texas has afforded the opportunity to examine the complex direct and indirect multitrophic interactions occurring within the citrus phyllosphere. For the past several decades, A. woglumi populations of citrus in the LRGV generally have been regulated at subeconomic densities by an introduced parasite complex that includes two parasitic wasp species: A. hesperidum (Hymenoptera: Platygasteridae) and E. opulenta (Hymenoptera: Encyrtidae) [24, 29, 59]. Although causal factors were not determined, the most recent outbreak (2003) of A. woglumi, the third since 1983, was particularly severe in western regions of the LRGV and illustrates the dynamic nature of this host–parasite relationship. The large increase in A. woglumi population numbers resulted in extremely heavy deposits of sooty mold on citrus over a large area, which caused substantial crop losses for producers. The direct and indirect impacts of parasite populations on size of A. woglumi populations, fungal community size and structure, and plant health emphasize the multitrophic nature of this complex system. In orchards, the eventual population recovery of the aforementioned parasites resulted in a general decline of A. woglumi populations by mid-2005 (K. R. Summy, personal observation). Insect honeydew provides a good growth substrate for various saprophytic fungi [3, 8]. The primary sooty mold species found on citrus has been classically referred to as Capnodium citri and has been a useful moniker in the horticultural but not the mycological sense. However, Reynolds [50] shows that this is a complex of species that are taxonomically included in the Capnodiaceae (Capnodiales) and represent Caldariomyces, Polychaeton, Anten- Table 4 Simpson’s inverse diversity index (1/DS) and Shannon’s (H) diversity index for various levels of sooty mold (VSMR), citrus blackfly (CBF), and parasite exit holes (PEH) on C. paradisi phyllospheres Diversity indicesa VSMR 0 1 2 3 CBF 1 2 3 PEH 0 1 2 3 Adults 0 1 a Richnessb 1/DS ± SD H′ ± SD 9 11 16 18 4.49±0.13 3.31±0.16 2.60±0.08 2.76±0.02 1.64±0.04 1.45±0.03 1.19±0.04 1.32±0.01 18 12 20 2.93±0.04 2.69±0.08 2.67±0.10 1.30±0.02 1.24±0.04 1.44±0.03 20 22 17 8 2.82±0.09 2.23±0.03 3.79±0.46 2.55±0.19 1.40±0.02 1.16±0.01 1.68±0.15 1.25±0.07 20 21 3.09±0.04 2.61±0.06 1.44±0.01 1.33±0.02 Simpson’s inverse diversity index, 1/DS; Shannon’s diversity index, H′ (±standard deviation, SD) b Species richness: number of fungal groups out of the total 22 isolated Phyllosphere Fungal Communities nariella, and Chaetobolisia. In addition to the C. citri “complex,” a number of other highly melanized forms may coexist on insect honeydew including Fumago, Limacinia, Morfea, Scorias, and Tripospermum [22, 50]. In addition to the classical “sooty mold” fungi, a number of different Fusarium spp. were recovered from molded leaves including F. solani and F. oxysporum as well as several isolates that primarily produced conidia in long chains on monophialides (and polyphialides) that represent members of the G. fujikuroi complex of mating type species, e.g., F. moniliforme sensu lato [58]. Further studies need to be conducted in order to fully characterize the Fusarium communities on grapefruit leaves. A number of yeast species have been reported from Citrus spp. in the past [22, 23, 61, 63]. The results of this study indicated that yeasts (primarily red and white types) and dimorphic forms represented over 5% of the mycobiota, overall. However, yeast populations dramatically declined (relative to filamentous forms) as VSMR increased in orchards (Fig. 1; Table 3). The observed trend suggests that support of a naturally high yeast population (relative to that of filamentous fungi) may be a potential route of biocontrol for sooty mold in citrus [35]. High yeast populations have been associated with biocontrol of post-harvest fruit rots in citrus although the mechanism has not been fully elucidated [42]. Further phyllosphere yeasts, such as Sporobolomyces spp. and Cryptococcus spp. on wheat leaves, as well as uncharacterized, heterogeneous yeast populations on cotton leaves and bolls, have the ability to grow on many of the sugars that are typically found in insect honeydew, such as fructose, glucose, melezitose, sucrose, and trehalose [18, 21]. Saprophytic yeasts are common residents of leaf surfaces and are an indicator of plant health. In fact, Dik et al. [17] showed that accumulation of honeydew on wheat flag leaves stimulates necrotrophic pathogenesis, and conversely, the presence of honeydew-consuming saprophytic yeasts on the phylloplane may result in an overall increase in wheat yield. These potential interactions within a trophic level (microbial community on the phylloplane) emphasize the complexity of interactions with myriad direct and indirect effects that may occur within the phyllosphere. The accumulation of sooty mold (regardless of fungal species) may have a number of direct and indirect effects on the plant in question. Accumulation of sooty mold interferes with light absorption by the leaf and thus reduces photosynthetic efficiency. This problem has been reported in such crops as zucchini, avocado, and “ma kiang” trees [13, 15, 45, 52]. Recent spectroradiometric experiments with grapefruit have shown decreased red and blue light absorption in leaves that have been covered with sooty mold for a long period of time [39]. While Fusarium and Aspergillus fruit rots do occur, they are rare but may have important direct effects on citrus that lead to economic losses for growers. Fusarium spp. are common on citrus leaves and fruit. For example, a number of common Fusarium spp. are associated with fruit rots and have been routinely isolated from leaf surfaces in this study, such as F. oxysporum that causes a fusarium wilt in citrus. In the early 20th century, Ghatak [26] indicated that at least two strains of F. moniliforme sensu lato were responsible for post-harvest rot in storage conditions. The isolation of P. digitatum and P. italicum is important as these penicillia are responsible for “green mold” and “blue mold” of citrus fruit, and A. niger will cause a “black mold rot” of citrus [61]. Although these diseases are considerations in the postharvest setting for fruit, the inoculum may originate from other plant parts (such as sooty-molded leaves) and be delivered to the fruit surface and stem-end during the harvesting process. An important environmental factor that may affect fungal community structure is UV irradiation. Interestingly, several moniliaceous hyphomycetes were isolated in this study (Table 3). It is probable that these fungi either are protected by the surrounding melanized species or were isolated from leaves that were taken from lower points in the tree canopy. Hyphal cell wall melanization is a beneficial adaptation that enhances phyllocompetency in fungi [6, 11, 33]. Further, melanization has been shown to lend protection from UV, heat, and oxidative damage [12, 20, 25, 40, 49]. In addition, melanin is required for pathogenic mechanisms (e.g., infection structure development) in some fungi, and some derivatives (e.g., 1,8dihydroxyquinone) may have phytotoxic properties [16, 31, 32, 51, 64]. Several authors have recovered moniliaceous (Fusarium, Aspergillus, and Penicillium spp.) and dematiaceous hyphomycetes (Alternaria, Aureobasidium, Cladosporium, Drechslera, Epicoccum, Stemphylium, and Ulocladium spp.) from citrus leaf surfaces [5, 22, 23, 36]. However, none of the referenced work establishes that these fungi are “residents” of the phyllosphere. It is possible that some of the fungi isolated in this study do not reside on the phyllosphere, but are deposited there through dissemination by airborne spores. For example, airborne conidia of Alternaria spp. have been routinely trapped in citrus canopies in Florida [62]. Otherwise, very little aeromycology work has been performed to characterize the airborne fungi within the citrus canopy. Previous work has indicated that microbial communities are “dynamic” and that “turnover in species composition” naturally occurs on leaves in an orchard [4, 38]. One can therefore hypothesize that such natural phyllosphere fungal community transitions, like those observed in this study, could be influenced, perhaps disproportionately, by excessive feeding activities and honeydew deposition of insects such as Aleurocanthus woglumi. In turn, failure to maintain J. L. Perez et al. population equilibrium of the pest by parasites, such as Amitus and Encarsia, will clearly result in a carbon source inundation of the natural phyllosphere fungal community. This multitrophic upset leads to domination of the leaf surface landscape by saprophytic and potentially pathogenic filamentous fungi. Subsequently, this partial “invasive weed effect” results in limitations of the normal diversity that should model the healthy phyllosphere. Often, the effect of an invasive weed on a macrobiotic landscape is to decrease species diversity, decrease richness, distort eveness, and promote dominance by one to a few species. The decline in fungal community diversity as a result of increased dominance of Capnodiales and Hypocreales observed in this study is consistent with this idea (Table 4). The direction of a trophic cascade (top–down versus bottom–up) for similarly structured food chains may depend on the specific species involved and the complex nature of interactions among those species. Other studies investigating insect food chains with fungi at the base have suggested bottom–up control of parasitoids by endophytic fungi [28, 46]. Overall, our results suggest that the interaction between the parasitoid and the fungal phyllosphere community may represent a top–down trophic cascade with the parasite having a negative impact on fungal community abundance and diversity. Clearly, to confirm this relationship between phyllosphere diversity and parasitoid abundance, experimental work altering parasitoid levels will be required. However, to our knowledge, the present study is the first to examine the effects of indirect interactions among trophic levels on fungal communities in the phyllosphere. Acknowledgements The authors would like to thank Saulina Orizaga for assistance in processing leaf samples for mycobiotic data. Thanks to Mani Skaria and John DaGraça (Texas A&M University—Kingsville Citrus Center, Weslaco, Texas) for helpful comments throughout the work and assistance in reviewing the final manuscript. The authors also thank Texas Citrus Mutual (Mission, Texas) in assistance in collecting visual insect and sooty mold data from citrus producers. This paper is Contribution no. 09-087-J from the Kansas Agricultural Experiment Station, Manhattan. Also, this paper is Publication no. CSS 2008-02 of the UTPA Center for Subtropical Studies. References 1. Alvarez MG (1976) Primer catalogo de enfermedades de plantas Mexicanas. Fitofilo 71:1–169 2. Ancisco JR, French JV, Skaria M, Sauls JW, Holloway R (2002) IPM in Texas Citrus. Texas Cooperative Extension, The Texas A&M University System, Publication B-6121 3. Andrews JH (1982) Biological control in the phyllosphere. Ann Rev Phytopathol 30:603–635 4. Andrews JH, Kinkel LL, Berbee FM, Nordheim EV (1987) Fungi, leaves, and the theory of island biogeography. Microb Ecol 14:277–290 5. Araújo WL, Maccheroni W, Aguilar-Vildoso CI, Barrosa PA, Saridakis HO, Azevedo JL (2001) Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Can J Microbiol 47:229–236 6. Bell AA, Wheeler MH (1986) Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24:411–451 7. Beyer HL, Merill EH, Varley N, Boyce MS (2007) Willow on Yellowstone’s northern range: Evidence for a trophic cascade? Ecol Appl 17:1563–1571 8. Blakeman JP, Fokkema NJ (1982) Potential for biocontrol of plant diseases on the phylloplane. Ann Rev Phytopathol 20:167–192 9. Booth C (1977) Fusarium: laboratory guide to the identification of the major species. Commonwealth Mycological Institute, Kew 10. Bovi MLA, Vilela de Resende MD, Saes LA, Uzzo RP (2004) Genetic analysis for sooty mold resistance and heart of palm yield in Archontophoenix. Sci Agric 61:178–184 11. Butler MJ, Day AW (1998) Fungal melanins: a review. Can J Microbiol 44:1115–1136 12. Butler MJ, Lazarovits G, Higgins BG, Lachance MA (1989) Identification of a black yeast isolated from oak bark belonging to the genus Phaeococcomyces sp.: analysis of melanin produced by the yeast. Can J Microbiol 35:728–734 13. Chen J, McAuslane HJ, Carle RB, Webb SE (2004) Impact of Bemesia argentifolii (Homoptera: Auchenorrhyncha: Aleyrodidae) infestation and squash silverleaf disorder on zucchini yield and quality. J Econ Entomol 97:2083–2094 14. Colwell RK (2005) EstimateS: statistical estimation of species richness and shared species from samples. Version 7.5. User’s Guide and Application published at: http://purl.oclc.org/estimates 15. Davidson EW, Segura BJ, Steele T, Hendrix DL (1994) Microorganisms influence the composition of honeydew produced by the silverleaf whitefly, Bemesia argentifolii. J Insect Physiol 40:1069–1076 16. DeJong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389:244–245 17. Dik AJ, Fokkema NJ, van Pelt JA (1991) Consumption of aphid honeydew, a wheat yield reduction factor, by phyllosphere yeasts under field conditions. Eur J Plant Pathol 97:209–232 18. Dik AJ, Fokkema NJ, van Pelt JA (1992) Influence of climatic and nutritional factors on yeast population dynamics in the phyllosphere of wheat. Microb Ecol 23:41–52 19. Drees BM, Jackman JA (1998) A field guide to common Texas insects. Gulf Publishing, Lanham 20. Durrell LW (1964) The composition and structures of walls of dark fungus spores. Mycopathol Mycol Appl 23:339–345 21. Elliot VJ (2002) Ability of indigenous yeasts from aerial plant surfaces to degrade sugars from insect honeydew. J Cotton Sci 6:60–67 22. Farr DF, Bills GF, Chamuris GP, Rossman AY (1989) Fungi on plants and plant products in the United States. APS Press, St. Paul 23. Fenn ME, Dunn PH, Durall DM (1989) Effects of ozone and sulfur dioxide on phyllosphere fungi from three tree species. Appl Environ Microbiol 55:412–418 24. French JV, Meagher RL, Esau KL (1990) Release of two parasitoid species for biological control of citrus blackfly in south Texas. J Rio Grande Valley Hort Soc 43:23–27 25. Gan EV, Faberman HF, Menon IA (1976) Electron-transfer properties of melanin. Arch Biochem Biophys 173:666–672 26. Ghatak PN (1938) Investigations on orange rot in storage: I. Orange rot due to two strains of Fusarium moniliforme Sheldon. Ind Bot Soc J 17:141–148 27. Gullan PJ, Martin JH (2002) Sternorrhyncha (jumping plant lice, whiteflies, aphids, and scale insects). In: Encyclopedia of insects. Academic Press/Elsevier Science, New York 28. Härri SA, Krauss J, Muller CB (2008) Trophic cascades initiated by fungal plant endosymbionts impair reproductive performance of parasitoids in the second generation. Oecologia 157:399–407 Phyllosphere Fungal Communities 29. Hart W, Meyerdirk D, Sanchez M, Stone W, Rhode R (1978) Development of a trap for the citrus blackfly, Aleurocanthus woglumi Ashby. Southwest Entomol 3:219–225 30. Hill TCJ, Walsh KA, Harris JA, Moffett BF (2003) Using ecological diversity measures with bacterial communities. FEMS Microbiol Ecol 43:1–11 31. Howard RJ, Ferrari MA (1989) Role of melanin in appressorium function. Exp Mycol 13:403–418 32. Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substances by a fungus employing enormous turgor pressures. Proc Natl Acad Sci 88:11281–11284 33. Jacobsen ES (2000) Pathogenic roles for fungal melanin. Clin Microbiol Rev 13:708–717 34. James DG, Stevens MM, O’Malley KJ, Faulder RJ (1999) Ant foraging reduces the abundance of beneficial and incidental arthropods in citrus canopies. Biol Control 14:121–126 35. Kohl J, Fokkema NJ (1998) Strategies for biological control of necrotrophic fungal pathogens. In: Boland GJ, Kuykendall LD (eds) Plant microbe interactions and biological control. Marcel Dekker, New York, pp 49–88 36. Koizumi M, Kuhara S (1984) Yeast-type organisms as citrus pseudo greasy spot (Nise-Ohan-Byo) causal agent. Ann Phytopathol Soc Japan 50:620–627 37. Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell Publishing, Ames 38. Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883 39. Little CR, Summy KR (2005) Detection of stressors in glasshouse crops using color infrared imagery: I. Damage caused by fungal pathogens and sooty mold. In: Proceedings of the 20th Biennial Workshop on Color Photography & Videography and Airborne Imaging for Resource Assessment. American Society for Photogrammetry and Remote Sensing, Weslaco, Texas, 12 pp 40. Lukiewicz S, Reska K, Matusak Z (1980) Simultaneous electrochemical-electron spin resonance (SEESR) studies on natural and synthetic melanins. Bioelectrochem Bioenerg 7:153–165 41. McGuire JU, Crandall BS (1967) Survey of insect pests and plant diseases of selected food crops of Mexico, Central America and Panama. Int. Agric. Dev. Serv., ARS, USDA-AID 42. McLaughlin RJ, Wilson CL, Chalutz E, Kurtzman CP, Fett WF, Osman SF (1990) Characterization and reclassification of yeasts used for biological control of post-harvest diseases of fruits and vegetables. Appl Environ Microbiol 56:3583–3586 43. Muhlenberg E, Stadler B (2005) Effects of altitude on aphidmediated processes in the canopy of Norway spruce. Agric Forest Entomol 7:133–143 44. Pace ML, Cole JJ, Carpenter SR, Kitchell JF (1999) Trophic cascades revealed in diverse ecosystems. Trends Ecol Evol 14:483–488 45. Peña JE, Mohyuddin AI, Wysoki M (1998) A review of the pest management situation in mango agroecosystems. Phytoparasitica 26:1–20 46. Preszler RW, Gaylord ES, Boecklen WJ (1996) Reduced parasitism of a leaf-mining moth on trees with high infection frequencies of an endophytic fungus. Oecologia 108:159–166 View publication stats 47. Pretorius MC, Crous PW, Groenewald JZ, Braun U (2003) Phylogeny of some cercosporoid fungi from citrus. Sydowia 55:286–305 48. Ramirez C (1982) Manual and atlas of the penicillia. Elsevier Biomedical Press, New York 49. Rehnstrom AL, Free SJ (1997) The isolation and characterization of melanin-deficient mutants of Monilinia fructicola. Physiol Mol Plant Pathol 49:321–330 50. Reynolds DR (1999) Capnodium citri: the sooty mold fungi comprising the taxon concept. Mycopathologia 148:141–147 51. Robeson D, Stroebel G, Matsumoto GK, Fisher EL, Chen MH, Clardy J (1984) Alteichin: an unusual phytotoxin from Alternaria eichorniae, a fungal pathogen of water hyacinth. Experentia 40:1248–1250 52. Sanyong S, Amarakul V (2001) GIS application for plant diseases distribution on jack fruit, bael fruit, ma kiang trees in lower northern part of Thailand. In: Proceedings of the 22nd Asian Conference on Remote Sensing. Centre for Remote Imaging, Sensing and Processing (CRISP). Singapore Institute of Surveyors and Valuers (SISV), and Asian Association of Remote Sensing (AARS), Singapore, 4 pp 53. SAS Institute, Inc (1988a) SAS/STAT procedures guide, release 6.03 edition. SAS Institute, Inc, Cary 54. SAS Institute, Inc (1988b) SAS/STAT user’s guide, release 6.03 edition. SAS Institute, Inc, Cary 55. SPSS, Inc (2000) Systat 10 Statistics—Volume I, v. 10. SPSS, Inc, Chicago 56. Stadler B, Müller T (1996) Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst. Oecologia 108:1432–1439 57. Stadler B, Müller T (2000) Effects of aphids and moth caterpillars on epiphytic microorganisms in canopies of forest trees. Can J Forest Res 30:631–638 58. Summerell BA, Salleh B, Leslie JF (2003) A utilitarian approach to Fusarium identification. Plant Disease 87:117–128 59. Summy KR, Gilstrap FE, Hart WG (1985) Aleurocanthus woglumi [Hom.: Aleyrodidae] and Encarsia opulenta [Hym.: Encyrtidae]: density-dependent relationship between adult parasite aggregation and mortality of the host. Entomophaga 30:107–112 60. Texas Agricultural Experiment Station (1988) Texas plant disease handbook. Texas Agricultural Extention Service, The Texas A&M University System, Publication 2M-4-88, Revision 61. Timmer LW, Garnsey SM, Graham JH (2000a) Compendium of citrus diseases, 2nd edn. APS Press, St. Paul 62. Timmer LW, Darhower HM, Zitko SE, Peever TL, Ibáñez AM, Bushong PM (2000b) Environmental factors affecting severity of Alternaria brown spot of citrus and their potential use in timing fungicide applications. Plant Disease 84:638–643 63. Yang CH, Crowley DE, Borneman J, Keen NT (2001) Microbial phyllosphere populations are more complex than previously realized. Proc Natl Acad Sci 98:3889–3894 64. Yoder OC, Turgeon BG (1996) Molecular–genetic evaluation of fungal molecules for roles in pathogenesis to plants. J Genet 75:425–450