Chemistry of Cirsium and Carduus: a role in ecological risk assessment for biological control of weeds?

Prediction of host plant range and ecological impact of exotic phytophagous insects, such as insects for classical biological control of weeds, represents a major challenge. Recently, the flowerhead weevil (Rhinocyllus conicus Fröl.), introduced from Europe into North America to control exotic thistles (Carduus spp.), has become invasive. It feeds heavily on some, but not all species of native North American thistles (Cirsium spp.). We hypothesized that such non-target use among native plants could be better predicted by knowledge of characteristic chemical profiles of secondary compounds to supplement the results of host specificity testing. To evaluate this hypothesis, we reviewed the literature on the chemistry of Cirsium and Carduus thistles. We asked what compounds are known to be present, what is known about their biological activity, and whether such information on chemical profiles would have better predicted realized host range and ecological effects of R. conicus in North America. We found an extensive, but incomplete literature on the chemistry of true thistles. Two main patterns emerged. First, consistent chemical similarities and interesting differences occur among species of thistles. Second, variation occurs in biologically active groups of characteristic compounds, specifically flavonoids, sterols, alkaloids and phenolic acids, that are known to influence host plant acceptance, selection, and feeding by phytophagous insects. Surprisingly, sesquiterpene lactones, which are characterisitic in closely related Asteraceae, have not been extensively reported for Cirsium or Carduus. The minimal evidence on sesquiterpene lactones may reflect extraction methods vs. true absence. In summary, our review suggests further research on thistle chemistry in insect feeding is warranted. Also, since the exotic Canada thistle (Cirsium arvense) is an invasive thistle of current concern in North America, such research on mechanisms underlying host range expansion by exotic insects would be useful.     

Laboratory Trials of the Efficacy of the Crown Weevil Mortadelo horridus (Coleoptera: Curculionoidae) for the Biological Control of Slender Thistles Carduus pycnocephalus and C. tenuiflorus in Southwestern Australia

The hypothesis that larval crown weevil (Mortadelo horridus Alonso-Zarazaga and Sanchez-Ruiz) can suppress vegetative growth, seed set and viability in the winter annual slender thistles (Carduus pycnocephalus L. and C. tenuiflorus Curt.) was tested in laboratory gardens. Different densities (eight and 16) of M. horridus larvae and eggs were inoculated onto slender thistle rosettes between one and four times. Controls received no eggs or larvae. Larval feeding destroyed the apical meristems of treated plants, producing multi-crowned rosettes and significant declines in leaf area in both species, irrespective of the number of larvae. Treated thistles bolted at the same time and had a 4-fold increase in stem number compared to controls. The stems of control plants were up to one-third taller and thicker than attacked plants. Relative to controls, the leaves of treated plants had significantly fewer spines, decreased leaf area and decreased leaf perimeter, with less deeply lobed leaf margins. The total number of capitula produced by control and experimental groups was not significantly different, but consideration of fertile capitula only revealed a reduction in fertile capitula production following weevil attack. This was most marked in C. pycnocephalus. Control plants also produced significantly wider capitula. No control plants died, compared to 17 and 26% of the attacked plants of C. pycnocephalus and C. tenuiflorus, respectively. Implications for biocontrol of slender thistles are discussed.     

Neighbourhood of host plants influences oviposition decisions of a stem-boring weevil

The spatial arrangement of suitable host plants in the field may significantly constrain insects to find optimal hosts. Plant neighbours around a focal host plant can either lead to lower (associational resistance) or higher (associational susceptibility) herbivore loads. We tested whether the spatial arrangement of hosts of different suitability for the larval development of the shoot-base boring weevil Apion onopordi affects oviposition decisions in the field. Host plants in our study were healthy creeping thistles (Cirsium arvense; suboptimal hosts) and thistles infected by a rust pathogen (Puccinia punctiformis; optimal hosts). For analysis, we used nearest neighbour methods that disentangle the spatial distribution of organisms that are dependent on the position of other species (e.g. phytophagous insects and their host plants). Although theory predicts that the small-scale spatial infestation pattern can have major consequences for the population dynamics in insect–plant systems, field studies quantifying spatial pattern of phytophagous insects are rare.

The spatial arrangement of host plants clearly influenced oviposition pattern in A. onopordi. In contrast to previous studies, we demonstrated that not the rust infection itself determined if a plant was infested by weevils, but rather the density of rusted shoots within a certain neighbourhood. We found strong indications for associational susceptibility of healthy thistle shoots to weevil oviposition when growing in the neighbourhood of rusted thistles. Weevil-infested plants were spatially aggregated, indicating that A. onopordi is limited in its dispersal ability within patches. Other stem-boring insects on creeping thistle were affected in their oviposition decisions by other factors than A. onopordi. Thus, it may be difficult to find general rules for oviposition choice in phytophagous insects.

Our study showed that the spatial arrangement of host plants in the field critically determines oviposition choice and should thus be included as constraint in theories of optimal host selection.     

Exotic thistles increase native ant abundance through the maintenance of enhanced aphid populations

Exotic species change the structure and composition of invaded communities in multiple ways, but the sign of their impact on native species is still controversial. We evaluated the effects of the thistles Carduus thoermeri and Onopordum acanthium—two of the most abundant exotic plant species in disturbed areas of the Patagonian steppe—on the native tending ant assemblage. Exotic thistles showed an increased number of plants with aphids and had greater aphid density than native plants. Since native tending ants were present only in plants with aphids, their abundance was higher in infested thistles than in native plants. Path analyses confirmed that ant activity depended more on aphid density than on thistle traits. Our results suggest that the presence of exotic thistles in disturbed areas of NW Patagonia indirectly benefit the native ant assemblage through the maintenance of an increased aphid population. This illustrates how the impact of exotic on native species can depend on the ecological context.     

Endangered by laws: potential consequences of regulations against thistles on bumblebee conservation

Many bumblebee (Bombus) species are undergoing a strong decline in Europe due to, amongst other things, a decrease of food resources. While leguminous plants (Fabaceae) are considered to be one of the main pollen sources of bumblebees, thistles (Asteraceae tribe Cardueae) have been suggested to be important for male diet. Yet, several European countries apply strict regulations against thistles since they are considered to be one of the principal weeds in agricultural landscapes. Such regulations could impact bumblebee conservation through disruption of male diet and ecology. Here, we assess the male-depending importance of thistles for bumblebee species based on field observations across countries where a legal regulation against thistles is in effect. We ultimately aim to evaluate the potential consequences of these regulations on bumblebee conservation. Our results confirm that most floral visit observations of males occur on thistles (mainly Cirsium spp. and Carduus spp.) and some species are almost exclusively observed on them. Thistle removal is thus most likely a threat for bumblebees. Therefore, we advocate repealing the thistle removal acts to make way for alternative thistle regulations which reconcile biodiversity conservation and agricultural requirements.     

Hosts as habitats: faunal similarity of phytophagous insects between host plants

1. Published lists on the phytophages recorded on 86 cabbage plant species (Brassicaceae) and 30 thistle species (Cynaroideae) were used to investigate patterns in the faunal similarity of phytophages. This was done by calculating the Jaccard index and a standardised index of similarity between pairs of host species using presence/absence data. 2. The faunal similarity measured as Jaccard indices indicated that pairs of cabbage hosts share on average 36% of phytophagous species whereas pairs of thistle hosts share only 10%. 3. The faunal similarity between two host species increased with the taxonomic affinity of hosts. This increase was more pronounced in thistles than in cabbage plants. 4. Irrespective of the taxonomic affinity of hosts, in the cabbage plants the faunal similarity of endophages was lower than in ectophages. In contrast, in the thistles faunal similarity differed only between endophages and ectophages for hosts of the same genera. 5. Differences in the patterns of faunal similarity between the two host taxa may be due to idiosyncratic characteristics of the plant taxa, e.g. the unique chemical properties of the cabbage plants and the resource‐rich flower heads of thistles.     

Host recognition by Rhinocyllus conicus of floral scents from invasive and threatened thistles

One of the main obstacles of classical biological control is that biological control organisms cannot be recalled once they are released in nature. It is particularly true for the flowerhead weevil, Rhinocyllus conicus Frölich, which was released as a biological control organism for the invasive musk thistle, Carduus nutans L. (MT). While weevils successfully suppressed introduced populations of musk thistles and other invasive thistle species, non-target attacks have been reported on multiple native thistles including federally listed threatened and endangered (T&E) thistle species. To investigate the foraging behavior of female weevils on invasive and native thistles, we examined volatile organic compounds (VOCs) emitted from MT and a T&E plant species, Sacramento Mountains thistle, Cirsium vinaceum Wooton & Standley (SMT) in the Lincoln National Forest, New Mexico. We used a dynamic headspace volatile collection system and gas chromatography-mass spectrometry to compare volatile profiles between MT and SMT. Female weevils reacted to 7 electrophysiologically active chemical compounds in the blends based on gas chromatography-electroantennography. The behavioral response of female weevils was indifferent when VOCs from both thistles were offered in y-tube olfactometry experiments. Yet, they preferred VOCs collected from MT to purified air. The searching time of female weevils was longer to VOCs collected from SMT over controls. Investigating signals during the initial host recognition of released biological control organisms may open new opportunities to reduce non-target attacks on T&E plant species.

     

Spatial Segregation of Congeneric Invaders in Central Pennsylvania, USA

Carduus acanthoides and Carduus nutans (plumeless and musk thistles) are among the most noxious weeds in the United States of America, presenting a serious challenge in cropping and pasture systems. Unfortunately, a lack of detailed spatial distribution information hampers both our ability to understand the factors affecting their invasive success, and the effectiveness of monitoring and management efforts. To examine patterns of distribution and co-occurrence at a local level, we sampled a 5000 km² area of central Pennsylvania that cut a transect across known areas of C. acanthoides and C. nutans infestation. A number of potential environmental explanatory variables were recorded and analyzed to examine whether they correlated with observed species distribution patterns. Patterns of forest density and spatial aggregation of the thistles were the primary covariates that significantly impacted both species’ distributions. The survey established that the frequency of sightings for each species diminished as the ranges converged, with only brief overlap: the two species are strongly negatively correlated in space. Understanding environmental correlates of infestation and the pattern of spatial dissociation of these two invasive species is an important step towards an improved understanding of the mechanisms underlying their invasive potential, and hence towards effective weed control.     

Why close relatives make bad neighbours: phylogenetic conservatism in niche preferences and dispersal disproves Darwin's naturalization hypothesis in the thistle tribe

The number of exotic plant species that have been introduced into the United States far exceeds that of other groups of organisms, and many of these have become invasive. As in many regions of the globe, invasive members of the thistle tribe, Cardueae, are highly problematic in the California Floristic Province, an established biodiversity hotspot. While Darwin's naturalization hypothesis posits that plant invaders closely related to native species would be at a disadvantage, evidence has been found that introduced thistles more closely related to native species are more likely to become invasive. To elucidate the mechanisms behind this pattern, we modelled the ecological niches of thistle species present in the province and compared niche similarity between taxa and their evolutionary relatedness, using fossil‐calibrated molecular phylogenies of the tribe. The predicted niches of invasive species were found to have higher degrees of overlap with native species than noninvasive introduced species do, and pairwise niche distance was significantly correlated with phylogenetic distance, suggesting phylogenetic niche conservatism. Invasive thistles also displayed superior dispersal capabilities compared to noninvasive introduced species, and these capabilities exhibited a phylogenetic signal. By analysing the modelled ecological niches and dispersal capabilities of over a hundred thistle species, we demonstrate that exapted preferences to the invaded environment may explain why close exotic relatives may make bad neighbours in the thistle tribe.     

Fire‐induced colonization of a Flooding Pampa grassland by thistles: Remnant litter and interference effects

Abstract. Winter and spring burnings constitute a traditional management practice of the Flooding Pampa grasslands which are dominated by the tussock grass Paspalum quadrifarium. In addition to increasing the primary productivity and the nutrient quality of the regrowth, this burning favours the establishment of opportunistic species, especially the legume Lotus glaber and the thistles Cirsium vulgare and Carduus acanthoides. The aims of the present study were to assess the effects of burning and those of the remnant litter on L. glaber and thistle recruitment, as well as the effects of L. glaber density on the emergence, survival and flowering of thistles. Two field experiments were carried out: 1. A completely randomized factorial design with occurrence of L. glaber and fire was applied in a mature Paspalum stand; 2. A completely randomized design with four L. glaber seed densities oversown on a burned Paspalum stand, as the treatments. Thistle and L. glaber recruitment within the Paspalum stand was dependent on fire, but their emergence in burnt plots was reduced by the presence of remnant litter. Remnant litter and interference from L. glaber provide complementary mechanisms to reduce fire‐cued colonization by thistles. While seedling emergence of thistles was mostly affected by the presence of remnant litter, seedling survival depended on local density of L. glaber. By the end of the first post‐fire growing season, the survivorship of established thistles was linearly reduced with increasing sowing density and above‐ground biomass of L. glaber. The maintenance of a significant litter coverage and a high density of L. glaber in the pre‐fire seed bank, as well as the oversowing of L. glaber after fire, may be useful tools in fire management of Paspalum stands aimed to improve their forage value with reduced dependence on chemical control of weeds.     

Cryptic local adaptation of the herbivorous ladybird beetle Henosepilachna pustulosa in the ability to use the thistle Cirsium boreale

Local adaptation to different host plants is important in the diversification of phytophagous insects. Thus far, much evidence of the local adaptation of insects with respect to host use at the physiological level has been gathered from systems involving less mobile insects and/or divergent hosts such as plants belonging to different families or genera. On the other hand, the prevalence of such local adaptation of insects with moderate or high dispersal ability to the intraspecific variation of herbaceous hosts is largely unknown. In the present study, we examined the occurrence and degree of local adaptation of the herbivorous ladybird beetle Henosepilachna pustulosa (Kôno) (Coleoptera: Coccinellidae) to its primary host, the thistle Cirsium boreale Kitam. (Asteraceae), through reciprocal laboratory experiments using beetles and thistles from three locations with a range of approximately 200 km. Concerning the larval developmental ability, obvious patterns of local adaptation to the thistles from respective natal locations were detected, at least in some combinations of beetle populations. Similar tendencies were detected concerning adult feeding acceptance, although the statistical support was somewhat obscure. Overall, our results indicate that the degree of local adaptation of insect species with moderate dispersal ability to conspecific herbaceous hosts is occasionally as strong as that involving less mobile insects and/or heterospecific hosts, indicating the potential of such cryptic local adaptation to promote ecological/genetic differentiation of phytophagous insect populations.     

Indirect interaction between a fungal plant pathogen and a herbivorous beetle of the weed<Emphasis Type="Italic"> Cirsium arvense</Emphasis>

Interactions between plants and their natural enemies are well studied, but investigations on the indirect interactions between plant enemies that simultaneously exploit a host plant are rare. Yet these plant-mediated interactions are important because they may affect not only the impact of plant antagonists on plant survival but may also influence the performance of the other plant exploiters. This study focused on the indirect effects of a systemic infection of creeping thistle, [irsium arvense (L.) Scop., with the necrotrophic fungus Phoma destructiva (Plowr.) on the phytophagous leaf beetle Cassida rubiginosa Müller, by examining egg deposition, food plant choice, and larval and pupal performance of the beetle. Thus, the results give a broader view than most other studies of plant-mediated effects of a pathogen on a phytophagous insect. Since both the beetle and the fungus are considered as agents for the biological control of C. arvense, the results are also of interest for applied ecology. Potted plants of C. arvense were inoculated with a conidiospore suspension of P. destructiva to cause a systemic infection of the plants. In a cage experiment, ovipositing females of C. rubiginosa showed a significant preference for healthy thistles. In dual-choice tests, adults of C. rubiginosa preferred leaf discs from healthy thistles over those from Phoma-infected thistles. The beetles also consumed significantly more leaf tissue from healthy than from infected plants. Development time from freshly hatched larvae until pupation was significantly longer for larvae fed on infected leaves. The weight of last-instar larvae and pupae was lower, and larval and pupal mortality was higher when larvae had been fed with infected compared to healthy leaves. Thus, the combined use of both potential biological control agents may be of lowered efficiency because (1) C. rubiginosa avoided infected thistles for both egg deposition and adult feeding and (2) Phoma infection negatively affected larval development and increased larval and pupal mortality of the beetle.     

Host utilization of field-caged native and introduced thistle species by Rhinocyllus conicus

Rhinocyllus conicus Fr?elich was introduced from Europe into North America as a biological control agent of the exotic weed Carduus nutans L. Concern exists over the feeding of this weevil on at least 25 species of native Cirsium thistles. Beginning in 2008, cage studies isolating adults of R. conicus on buds and flower heads of all eight thistle species (native and introduced) recorded from Tennessee were conducted to test if R. conicus could use these species for reproduction and what impacts larval feeding of R. conicus may have on seed production. Larvae of R. conicus completed development in heads of the native species C. carolinianum (Walter) Fernald and Schubert. and C. horridulum Michaux, and significant reductions in seed numbers of both species occurred during 2008. Rhinocyllus conicus oviposited on both C. carolinianum and C. horridulum at significantly greater levels than the introduced species C. arvense (L.) Scopoli and C. vulgare (Savi) Tenore. Infested heads of C. carolinianum contained numbers of R. conicus per centimeter of plant head width similar to Ca. nutans in 2008, and both native species contained numbers of R. conicus per centimeter of plant head width similar to C. arvense and C. vulgare in 2009. Body length was similar between R. conicus reared on native thistles and its target host Ca. nutans. This report is the first documentation of R. conicus feeding and reproducing on C. carolinianum and C. horridulum. Although R. conicus has been observed only on introduced thistles in naturally occurring populations in this region, the utilization of C. carolinianum and C. horridulum as host species in controlled conditions warrants continued monitoring of field populations and further investigation into factors that may influence nontarget feeding in the future.     

Structure of the phytophagous insect fauna of Onopordum thistles in the northern Mediterranean basin

Thistles of the genus Onopordum (Asteraceae: Cardueae) have become serious weed problems in parts of Australia and North America following introduction from their native Eurasian ranges, and are the target of a biological control programme in Australia. This paper analyses the results of three separate surveys of insects feeding and breeding on thistles of the genus Onopordum in the Mediterranean region. Of the 129 species found feeding, 74 species also bred on these thistles. Endophages comprised 54% of the breeding insects, with species that fed in the capitula and within the rosette crown/root area predominating. The Coleoptera and Hemiptera were the dominant endophage and ectophage taxa, respectively. Differences in the Onopordum fauna were noted between host species and between geographic regions, but this was largely due to the generalist component of the fauna. Onopordum specialists showed little variability across regions or between different species within the host genus. On comparing the fauna of Onopordum with that of other thistle genera, it was suggested that plant-herbivore interactions between Onopordum and its fauna are less highly evolved, in that there is less host specialization and a lesser degree of niche partitioning. This appears to be related to the low rate of speciation and habitat specialization within the host genus itself. The potential of certain genus-specific insects as biological control agents for Onopordum spp. is discussed. It is argued that the absence of a high degree of niche specialization could favour successful biological control, as the agent would be capable of inflicting damage over a wider range of resource gradients.     

Phylogeny and ecological radiation of New World thistles (Cirsium,Cardueae - Compositae) based on ITS and ETS rDNA sequence data

Sequence data from a portion of the external transcribed spacer (ETS) and internal transcribed spacers (ITS-1 and ITS-2) of 18S-26S nuclear ribosomal DNA were used to resolve historical biogeography and ecology of true thistles (Cirsium, Cardueae, Compositae) in the New World. The 650 base-pair, 3' portion of the ETS examined here showed a level of variation across taxa similar to that of the ITS sequences included. A maximum-likelihood tree based on combined ETS and ITS sequences leads us to suggest that the New World species of true thistles constitute a major lineage, which in turn comprises several smaller lineages. A western North American lineage shows weak quartet-puzzling support, but includes a well-supported lineage of species endemic to the California Floristic Province. Comparisons of this Californian lineage with other neoendemic angiosperm groups of the region show that the Californian Cirsium lineage exhibits unusually high ecological diversity for a group displaying such low levels of rDNA sequence divergence across taxa. Similarly low levels of sequence divergence were found throughout the New World Cirsium lineage. These results indicate either that Cirsium underwent a rapid ecological radiation in North America, or that rDNA evolution in North American Cirsium has been highly conservative.     

A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans.

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA^Gln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA^Gln to Gln-tRNA^Gln and Asp-tRNA^Asn to Asn-tRNA^Asn. Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.     

内蒙古克氏针茅草原土壤异养呼吸对土壤温度和水分变化的响应

土壤异养呼吸在野外自然条件下除受温湿度影响外, 还受其他多种因子的综合影响, 很难利用野外观测数据确定土壤异养呼吸对温湿度变化的响应方程形式, 以及温湿度间是否存在交互作用。该研究在严格控制温湿度的条件下对内蒙古克氏针茅(Stipa krylovii)(西北针茅(Stipa sareptana var. krylovii))草原土样进行室内培养实验, 旨在解决上述问题。该研究的正交实验包括5个温度梯度(9、14、22、30、40 ℃)和5个湿度梯度(土壤持水力(water holding capacity, WHC)分别为20%、40%、60%、80%、100%)。室内培养实验持续71天, 土壤异养呼吸速率测定为2天(后期为1周)一次, 土壤可溶性有机碳和微生物生物量碳含量测定约为18天一次。研究结果显示: 土壤异养呼吸与温度呈显著正相关(p < 0.001)且温度间差异显著(p = 0.001), 呼吸温度敏感性(Q₁₀)与土壤水分含量呈正相关(p < 0.001); 呼吸与土壤水分二项式拟合效果较好, 在80% WHC时呼吸速率最大, 且最适湿度随温度上升而增加。土壤温度和水分的交互作用显著(p < 0.05), 土壤异养呼吸最适响应方程为lnRh = 0.914 + 0.098T + 0.046M + 0.001TM - 0.002T² - 0.001M² (Rh为异养呼吸, T为温度, M为湿度), 这说明加和形式的温湿度响应模型可能优于乘积形式。微生物生物量碳与土壤异养呼吸的相关性随培养时间发生变化, 土壤可溶性有机碳与土壤异养呼吸无显著相关(培养第20天除外), 原因可能是培养期间微生物死亡或群落结构改变导致微生物总体代谢活性的变化。     

Responses of soil heterotrophic respiration to changes in soil temperature and moisture in a Stipa krylovii grassland in Nei Mongol

土壤异养呼吸在野外自然条件下除受温湿度影响外, 还受其他多种因子的综合影响, 很难利用野外观测数据确定土壤异养呼吸对温湿度变化的响应方程形式, 以及温湿度间是否存在交互作用。该研究在严格控制温湿度的条件下对内蒙古克氏针茅(Stipa krylovii)(西北针茅(Stipa sareptana var. krylovii))草原土样进行室内培养实验, 旨在解决上述问题。该研究的正交实验包括5个温度梯度(9、14、22、30、40 ℃)和5个湿度梯度(土壤持水力(water holding capacity, WHC)分别为20%、40%、60%、80%、100%)。室内培养实验持续71天, 土壤异养呼吸速率测定为2天(后期为1周)一次, 土壤可溶性有机碳和微生物生物量碳含量测定约为18天一次。研究结果显示: 土壤异养呼吸与温度呈显著正相关(p < 0.001)且温度间差异显著(p = 0.001), 呼吸温度敏感性(Q₁₀)与土壤水分含量呈正相关(p < 0.001); 呼吸与土壤水分二项式拟合效果较好, 在80% WHC时呼吸速率最大, 且最适湿度随温度上升而增加。土壤温度和水分的交互作用显著(p < 0.05), 土壤异养呼吸最适响应方程为lnRh = 0.914 + 0.098T + 0.046M + 0.001TM - 0.002T² - 0.001M² (Rh为异养呼吸, T为温度, M为湿度), 这说明加和形式的温湿度响应模型可能优于乘积形式。微生物生物量碳与土壤异养呼吸的相关性随培养时间发生变化, 土壤可溶性有机碳与土壤异养呼吸无显著相关(培养第20天除外), 原因可能是培养期间微生物死亡或群落结构改变导致微生物总体代谢活性的变化。