Wild fungus gardens contained similar peptaibols, indicating their ecological relevance, consistent with peptaibol-producing Trichoderma being an opportunistic pathogen of T. septentrionalis fungus gardens . In our laboratory experiments, two of the Trichoderma fractions with the highest abundance of peptaibols, fractions D and E, induced some of the strongest T. septentrionalis weeding responses , and purified peptaibols also induced a weeding response similar to that of theTrichoderma extract . Given that the Trichoderma fractions and purified peptaibols were derived from different strains and represent a diversity in peptaibol composition, the similarities in the observed ant behavioral activity are thus likely to be unrelated to specific peptaibols but rather generally attributable to the peptaibol class of metabolites. Furthermore, the presence of peptaibols in environmental fungus gardens lends credence to their ecological relevance and together with the experimental data parallels the logic of Koch’s postulates, suggesting that peptaibols are likely produced during Trichoderma infections of ant fungus gardens and induce ant defensive behaviors in response. Peptaibols are produced by fungi in the order Hypocreales, especially by members of the mycoparasitic family Hypocreaceae, which contains both Trichoderma and Escovopsis, a specialized ant fungus garden pathogen . Peptaibols have been hypothesized to be important for mycoparasitism , which we show here can include infections of ant fungus gardens. Interestingly, the genes needed to synthesize peptaibols are encoded within the genome of Escovopsis , vertical farming systems for sale and Escovopsis has been shown to induce a strong ant weeding response in tropical leafcutting fungus-growing ants , similar to the T. septentrionalis weeding behaviors that we observed in response to Trichoderma and its peptaibols.
We therefore hypothesize that peptaibolinduced weeding behaviors are conserved among diverse fungus-growing ants and may reflect an ancient means of pathogen detection and defense. Future work should test if ant weeding intensity is directly correlated with pathogen load, virulence, peptaibol production, or other contributing environmental factors, and also compare the behavioral responses of diverse fungus-growing ant species to diverse fungal pathogens with varying levels of virulence and specialization toward ant fungus gardens, e.g., as in ref. 58. This study demonstrates how T. septentrionalis ants protect their cultivar mutualist from opportunistic Trichoderma pathogens by sensing and responding to peptaibols as specific molecular cues that induce an ant weeding response. These cues included two previously undescribed bioactive peptaibol metabolites that we identified in this study. Future research will investigate whether ants directly sense peptaibols or indirectly respond to an intermediate signal produced by the cultivar in response to peptaibols, in addition to characterizing other potential signaling molecules that are unlikely to be present in our Trichoderma extracts . In contrast to the canonical logic of host immune responses, in which hosts directly respond to infections, T. septentrionalis responses to peptaibol signaling molecules comprise an extended defense response whereby T. septentrionalis ants respond to infections of their cultivar mutualist. Such extended defense responses may be a widespread but poorly recognized mechanism that increases host health indirectly by preventing harm to their beneficial symbionts.Vineyard-fl oor management strategies, such as weed control and cover-cropping, have wide-ranging impacts both inside the vineyard, in terms of crop management and productivity, and outside the vineyard, in terms of runoff and sediment movement into streams and rivers.
The increasing importance of water-quality issues statewide, including in Monterey County where the Salinas River drains into the Monterey Bay National Marine Sanctuary, highlights the need for management strategies that limit environmental impacts. Growers are interested in alternative weed-control practices and cover crops, but they need information in order to balance benefits with the economic realities of wine-grape production. We established a 5-year experiment in a commercial vineyard in Monterey County with the intent of identifying effective practices that can be integrated into the cropping system without negatively affecting winegrape production. The vineyard floor consists of two zones: the rows, a 2- to 4-foot-wide swath underneath the vines, which are managed primarily to control weeds by herbicide applications or cultural practices ; and the middles, interspersed between the rows, which are vegetated by cover crops or resident vegetation in the dormant season, and are tilled or left untilled in spring. Growers manage weeds in rows to reduce competition for water, nutrients and light , and to prevent tall-statured weeds such as horse weed from growing or climbing into the canopy, where they interfere with harvest. Growers transitioning to more sustainable production systems need information on how management practices affect the physical properties, health, organic matter and water retention of soil. We monitored soil microbial activity for arbuscular mycorrhizal fungi and soil microbial biomass, since weed control and cover-cropping can affect populations of beneficial soil microbes in annual crops . Dormant-season cover crops in the middles minimize runoff from winter rains . Many California growers are also willing to plant cover crops because they protect soil from nutrient and sediment loss in winter storms , suppress weeds , harbor beneficial arthropods , enhance vine mineral nutrition and increase soil organic matter .
Competition between vines and cover crops for soil moisture in spring, when both are actively growing, can lead to severe water stress and reduce grape production . However, wine-grape production is distinct from other cropping systems because water stress may be imposed to enhance wine composition ; this practice has been studied mostly in high-rainfall regions of California. The vineyard production region of Monterey County, in contrast, has low rainfall , and growers must weigh the benefits of cover crops with the possible need to replace their water use with irrigation. In addition, growers must decide on the type of vegetation to utilize in the middles. Resident vegetation is cheap and generally easy to manage. Cover crops can provide specific benefits such as nitrogen fixation or high biomass production and vigorous roots . There are many choices for cover crops in vineyard systems, ranging from perennial and annual grasses, to legumes . Each species has strengths and weaknesses, as well as associated seed and management costs.Row weed control treatments were: cultivation, post-emergence weed control only and pre-emergence herbicide , followed by post-emergence herbicide applications . Cultivations and herbicide applications were timed according to grower practices and label rates. Cultivations were carried out every 4 to 6 weeks during the growing season using a Radius Weeder cultivator . The cultivator used a metal knife that ran 2 to 6 inches below the soil surface cutting weeds off in the vine row; it had a sensor that caused it to swing around vines. Pre-emergence herbicides were applied in winter with a standard weed sprayer, and postemergence herbicides were applied in spring through fall as needed with a Patchen Weedseeker light-activated sprayer . An early and late-maturing cereal were chosen for the cover-crop treatments; legumes were not considered due to aggravated gopher and weed problems. Cover-crop treatments in the middles were: no cover crop , earlier maturing ‘Merced’ rye and later maturing ‘Trios 102’ triticale . Cover crops were planted with a vineyard seed drill in a 32-inch-wide strip in the middle of 8-foot-wide rows just before the start of the rainy season in November 2000 to 2004 . They were mowed in spring to protect vines from frost, and both cover-crop species senesced by summer. Prior to planting cover crops each November, vertical farming equipment row middles were disked to incorporate the previous year’s cover crop and stubble and prepare a seedbed. Weed control and cover-crop treatments were arranged in a 3 x 3 splitblock design with three replicate blocks covering a total of 23 vineyard rows . Each block contained six vine rows and six adjacent middles. Weed control treatments were applied along the entire length of each vine row ; cover-crop treatments were established along one-third of each middle and were continuous across the main plot treatments in each block.
Each replicate main plot-by-subplot treatment combination included 100 vines.Soil compaction. Soil compaction was measured in the vine row in November or December 2003, 2004 and 2005 with a Field Scout Soil SC-900 compaction meter . Ten sites in each plot were sampled to a depth of 15 inches. Soil moisture. Soil water storage was evaluated from volumetric soil moisture measurements taken in-row and adjacent middles to a depth of 3.5 feet at 1-foot intervals using a neutron probe. The neutron probe readings were calibrated with volumetric moisture measured from undisturbed soil cores collected at the site. Rainfall and runoff. A tipping bucket rain gauge with an 8-inch-diameter collector was used to monitor daily and cumulative rainfall at the field site. Runoff was collected at the lower end of the plots into sumps measuring 16 inches in diameter by 5 feet deep. Each sump was equipped with a device constructed from a marine bilge pump, a float switch and flow meter, to automatically record the runoff volume from the plots during storm events. During the second and third years the sampling devices were modified to collect water samples for sediment and nutrient analysis. Vine mineral nutrition. One-hundred whole leaves opposite a fruit cluster were collected from each plot at flowering in May 2003, 2004 and 2005. Petioles were separated from leaf blades, and tissue was immediately dried at 140°F for 48 hours and then sent to the ANR Analytical Laboratory for nutrient analyses. Petiole and leaf-blade tissue samples were analyzed for nitrate , ammonium , nitrogen , phosphorus , potassium , sulfur , calcium , magnesium , boron , zinc , manganese , iron and copper . Soil mineral nutrition. Composited samples from 10 soil cores taken to a depth of 1 foot were collected from the vine rows and middles at flowering as described above. Samples were air dried and sent to the ANR Analytical Laboratory for analyses. Soil samples were analyzed for pH, organic matter, cation exchange capacity , nitrate, Olsen-phosphorus, potassium, calcium, magnesium, sodium , chloride , boron and zinc. Soil microbial biomass. Due to the limited capacity of the laboratory, microbial biomass assays were conducted on selected treatments. Ten soil cores were collected to a depth of 1 foot and then composite samples were made from each replicate of the pre-emergence and cultivation weed-control treatments and the adjacent middles of the ‘Merced’ rye and bare treatments. Samples were collected about four times each year from November 2001 to November 2005 for a total of 14 sets of samples. Soil samples were immediately placed on ice and taken to the laboratory for soil microbial biomass carbon analysis according Vance et al. . Mycorrhizae. Roots were collected, stained and examined as previously reported on April 16, 2003, May 3, 2004, and June 2, 2005. Grape yield, fruit quality and vine growth. Fruit weight and cluster number were determined by individually harvesting 20 vines per subplot. Prior to harvest a 200-berry sample was collected from each subplot for berry weight and fruit composition. Berries were macerated in a blender and the filtered juice analyzed for soluble solids as Brix using a hand-held, temperature compensating refractometer. Juice pH was measured by pH meter and titratable acidity by titration with a 0.133 normal sodium hydroxide to an 8.20 pH endpoint. At dormancy, shoot number and pruning weights were measured from the same 20 vines. Statistical analysis. Analyses of variance were used to test the effects of cover crop, weed control and year on the vine, soil and microbial parameters, according to a split-block ANOVA model in SAS . Cover crop, weed control, year and their interactions were treated as fixed effects. The main and interactive effects of block were treated as random effects. Year was treated as a repeated measure. When necessary, data were log-transformed to meet the assumption of normality for ANOVA, although untransformed or reversetransformed means are presented. Changes in soil moisture among treatments during the winter and the irrigation seasons were determined from significant treatment-date interactions.We conducted evaluations with a penetrometer each fall to determine the impact of weed-control treatments on soil compaction. Soil compaction was not significantly different at any depth in 2003 . However, in 2004 and 2005 soil compaction began to increase in the cultivation treatment compared to the other two weed-control treatments. In 2004, soil compaction at the 4- to 7-inch depth was significantly greater in the cultivation treatment compared to the standard treatment , but not more so than in the post-emergence treatment . In 2005, the cultivation treatment had significantly greater soil compaction at the 4- to 7-inch depth than both the post emergence and standard weed-control treatments . At the 8- to 11-inch depth, soil compaction was significantly greater than the standard treatment , but not greater than in the post-emergence treatment .