Ray blight of pyrethrum in Australia: a review of the current status and future opportunities

Ray blight caused by Stagonosporopsis tanaceti is one of the most important diseases of pyrethrum (Tanacetum cinerariifolium), a perennial herbaceous plant cultivated for the extraction of insecticidal pyrethrins in Australia. The disease is responsible for complete yield loss in severe outbreaks. Infected seed is considered as the principal source of S. tanaceti. Infection hyphae remain only in the seed coat and not in the embryo, resulting in pre- and post-emergence death of seedlings and latent infection. Therefore, quantification of the level of infection by S. tanaceti within seed using a qPCR assay is important for efficient management of the disease. Stagonosporopsis tanaceti completes its life cycle within 12 days after leaf infection through production of pycnidia and can infect every tissue of the pyrethrum plant except the vascular and root tissues. Ray blight epidemics occur in pyrethrum fields through splash dispersal of pycnidiospores between adjacent plants. Besides steam sterilization, thiabendazole/thiram and fludioxonil are effective seed-treating chemicals in controlling S. tanaceti before planting begins. Ray blight is currently managed in the field through the foliar application of strobilurin fungicides in the first 1–2 years of crop establishment. Later on, difenoconazole and multisite specific fungicides in the next 2–3 years during early spring successfully reduce ray blight infestation. Avoiding development of resistance to fungicides will require more sustainable management of ray blight including the development and deployment of resistant cultivars.     

A new anthracnose disease of pyrethrum caused by Colletotrichum tanaceti sp. nov

A new pathogen of pyrethrum (Tanacetum cinerariifolium) causing anthracnose was described as Colletotrichum tanaceti based on morphological characteristics and a four‐gene phylogeny consisting of rDNA‐ITS, β‐tubulin (TUB2), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and actin (ACT) gene sequences. The fungus produced perithecia in culture, requiring an opposite mating type isolate in a heterothallic manner. The initial infection strategy on pyrethrum leaves involved the formation of appressoria followed by production of multilobed infection vesicles in the epidermal cells. Infection and colonization then proceeded through thinner secondary hyphae, which resulted in the initial production of water‐soaked lesions followed by black necrotic lesions. The infection process was suggestive of a hemibiotrophic infection strategy. Moreover, phylogenetic analysis clearly showed that C. destructivum, C. higginsianum and C. panacicola were separate species that also had similar intracellular hemibiotrophic infection strategies as C. tanaceti, which all clustered in the C. destructivum complex. Colletotrichum spp. were detected at 1% incidence in seed of 1 of 19 seed lines, indicating the potential for seed as a source of inoculum into crops. Colletotrichum tanaceti was detected in leaf lesions from 11 of 24 pyrethrum fields surveyed between April and July 2012, at a frequency of 1·3–25·0% of lesions. Anthracnose probably contributes to the complex of foliar diseases reducing green leaf area in pyrethrum fields in Australia.     

TaqMan PCR assay for detection and quantification of <Emphasis Type="Italic">Stagonosporopsis tanaceti</Emphasis> in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (C_t) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = ?0.999, P < 0.001) between the C_t value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.     

Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.     

Seed and pollen transmission of <Emphasis Type="Italic">Apple latent spherical virus</Emphasis> in apple

To examine whether Apple latent spherical virus (ALSV) has spread among apple trees in an orchard, we surveyed 21 apple trees surrounding two ALSV-infected trees for virus infection using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). None of the 21 trees were infected, indicating that ALSV has not spread from the infected trees to the neighboring apple trees since it was first detected in 1984. We analyzed seed embryos and seedlings derived from infected trees and detected ALSV in 10 of 223 seed embryos (4.5%) and 10 of 227 seedlings (4.4%). From these results, we conclude that ALSV is seed-transmitted at a rate of ca. 4.5% in apple. We also analyzed seed embryos and seedlings from uninfected apple trees that were hand-pollinated with pollen from infected trees. We detected ALSV in only 1 of 260 seed embryos and in none of the 227 apple seedlings. This result indicated that the seed transmission rate via infected pollen is only 0–0.38%. In situ hybridization analysis of ALSV-infected apple flower buds showed that ALSV was present inside almost all pollen grains and in all ovary and ovule tissues, including the embryo sac and inner integument.     

Tan spot of pyrethrum is caused by a Didymella species complex

Tan spot is a disease of pyrethrum (Tanacetum cinerariifolium) in Australia. Recent increases in the severity and incidence of the disease have prompted a re‐evaluation of the pathogen, originally described as Microsphaeropsis tanaceti, including its phylogenetic relationships and morphology. Nucleotide comparison of partial sequences of the nuclear ribosomal internal transcribed spacer, β‐tubulin, large subunit 28S nrDNA (LSU), actin and glyceraldehyde‐3‐phosphate dehydrogenase loci identified two distinct haplotypes within the species. Haplotype differentiation was consistent for each locus, except for the LSU, within which sequences were identical across all isolates. Morphological variation, especially culture pigmentation and conidial size, consistently supported the phylogenetic data distinguishing two haplotypes. Phylogenetic comparisons of M. tanaceti incorporating 98 Didymellaceae species did not associate the M. tanaceti haplotypes with the genus Microsphaeropsis. The two M. tanaceti haplotypes were closely related, and clustered in the Didymella sensu stricto clade. Based on these phylogenetic results, supported by their distinct morphology and cultural characteristics, the two haplotypes of M. tanaceti are reclassified as two species of Didymella, namely D. rosea and D. tanaceti. The implications of two closely related species causing tan spot of pyrethrum are discussed.     

Evidence for seed transmission and symptomless growth of Ramularia collo‐cygni in barley (Hordeum vulgare)

Ramularia collo‐cygni (Rcc) is becoming an increasing problem for barley growers across Europe. However, the life cycle of the pathogen is only slowly being elucidated. In this study, Rcc DNA was detected in a number of harvested seed samples from 1999 to 2010, with mean levels peaking in winter barley samples in 2009. A number of experiments were carried out to determine whether the pathogen could move from barley seed to seedlings, and also from seed through the developing plant and into the subsequent generation of seed, both in controlled experiments and in field trials. Results from testing of seed indicated that the fungus is widespread at the end of the growing season in harvested grain samples and can be transmitted to developing plants from infected seed stock. Examination of infected seedlings did not reveal the presence of spores but fungal structures were found within the leaf. The location of the fungus within seed was examined, with Rcc DNA found in both embryo and non‐embryo tissue. The implications for barley production of the pathogen being seedborne are discussed.     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.     

Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post‐flowering stress. Overseas reports of M. phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperature × soil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M. phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130 mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M. phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.     

Tan spot: a new disease of pyrethrum caused by Microsphaeropsis tanaceti sp. nov.

The isolation frequency of Microsphaeropsis sp. in spring in association with necrotic lesions on leaves in Tasmanian pyrethrum (Tanacetum cinerariifolium) fields has increased substantially since first identification in 2001. Examination of morphological features and sequencing of the internal transcribed spacer region (ITS) resulted in the identification of a new species, herein described as Microsphaeropsis tanaceti sp. nov. The pathogenicity of three M. tanaceti isolates to two pyrethrum cultivars was confirmed by inoculating glasshouse‐grown plants in three experiments. No significant differences in the susceptibility of the two cultivars to infection by M. tanaceti were found. Symptoms were tan‐coloured spots which coalesced around the margins of the leaves. Therefore, the name ‘tan spot’ is proposed for this new disease of pyrethrum. The sensitivity of seven M. tanaceti isolates to difenoconazole and azoxystrobin, commonly used fungicides for the management of foliar diseases in spring, was assessed under in vitro conditions. Sensitivity testing for difenoconazole was conducted using a mycelial growth assay on potato dextrose agar, whilst testing for sensitivity to azoxystrobin used a conidial germination assay on water agar. Microsphaeropsis tanaceti was found to be more sensitive to azoxystrobin than difenoconazole, with complete inhibition of conidial germination at concentrations above 0·625 µg a.i. mL^?1. By comparison, concentrations of 50 µg a.i. difenoconazole mL^?1 or greater were required for significant inhibition of mycelial growth. It therefore appears likely that there is currently some control of tan spot as a result of the use of azoxystrobin and to a lesser extent, difenoconazole, for the control of other diseases.     

Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas)

Sweet potato leaf curl virus (SPLCV) infects sweet potato and is a member of the family Geminiviridae (genus Begomovirus). SPLCV transmission occurs from plant to plant mostly via vegetative propagation as well as by the insect vector Bemisia tabaci. When sweet potato seeds were planted and cultivated in a whitefly‐free greenhouse, some sweet potato plants started to show SPLCV‐specific symptoms. SPLCV was detected by PCR from all leaves and floral tissues that showed leaf curl disease symptoms. More than 70% of the seeds harvested from SPLCV‐infected sweet potato plants tested positive for SPLCV. SPLCV was also identified from dissected endosperm and embryos. The transmission level of SPLCV from seeds to seedlings was up to 15%. Southern blot hybridization showed SPLCV‐specific single‐ and double‐stranded DNAs in seedlings germinated from SPLCV‐infected seeds. Taken altogether, the results show that SPLCV in plants of the tested sweet potato cultivars can be transmitted via seeds and SPLCV DNA can replicate in developing seedlings. This is the first seed transmission report of SPLCV in sweet potato plants and also, to the authors' knowledge, the first report of seed transmission for any geminivirus.     

The Relationship Between Wheat Seed Weight, Infection by Fusarium culmorum or Microdochium nivale, Germination and Seedling Disease

The distribution of seeds by weight for three lots of winter wheat cv. Avalon infected by Fusarium culmorum and three lots of winter wheat cv. Riband infected by Microdochium nivale was determined. The distribution of infected seeds within each seed lot was then determined by isolating F. culmorum from seeds on moist filter paper and M. nivale from seeds on potato dextrose agar. The distribution of M. nivale infected seeds between seeds of different weight was similar to that of the seed lot as a whole, whereas the distribution of F. culmorum was greater in light seeds than heavy seeds. The percentage germination of infected seeds decreased with seed weight. A similar situation was found with respect to seedling emergence in compost for F. culmorum infected seeds. However, with M. nivale infection, similar numbers of seedlings emerged from both light and heavy infected seeds. Seed treatment with guazatine increased seedling emergence for both light and heavy seed infected by M. nivale. However, seedling emergence from F. culmorum infected seed was poor even following treatment with guazatine. Poor emergence was most evident from light seed.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Comparative microscopic and molecular analysis of Thatcher near‐isogenic lines with wheat leaf rust resistance genes Lr2a,Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races

Thatcher near‐isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticina‐ and wheat‐specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P.  triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45·5–51·5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.     

Seed transmission of maize downy mildew (Peronosclerospora sorghi) in Nigeria

In an area of Nigeria where downy mildew of maize is present, histological assessment of maize seed revealed the presence of mycelium and oospores of Peronosclerospora sorghi in the kernels. Seed transmission of downy mildew of maize was demonstrated when grain purchased at local markets gave mean seedling infection rates of 12·3% (untreated seeds) and 10·0% (in metalaxyl-treated seeds) within 7 days of emergence, after storage in a desiccator for 30 days. When untreated seeds taken from nubbin ears of systemically infected plants from four states in southern Nigeria were planted at 9 days (17–22% moisture content) and 27 days (9–22% moisture content) after harvest, 20·0% infected seedlings resulted in both trials. Seeds from Borno state in northern Nigeria had 26·6% systemic seedling infection after 9 months of storage at 11% moisture content. When seeds harvested from maize plants inoculated with P. sorghi through silks were examined histologically, hyphae of P. sorghi were observed mostly in the scutellum of the embryo. Transmission of disease to seedlings was observed when the silk-inoculated seeds (9% moisture content) were planted in pots in a greenhouse; however, no disease transmission was observed when such seeds were planted in the field. The epidemiological significance of seed transmission is discussed with particular reference to survival of inoculum and development of epidemics. Also noteworthy is the overall significance of seed transmission in Nigeria, where the major source of seed is that saved by farmers from their grain crop, occasionally supplemented by seed bought from the local market.     

Silicon induced resistance in cucumber plants against Pythium ultimum

The effect of the amendment of nutrient solutions with soluble potassium silicate on the response of cucumber (cv. Corona) root and hypocotyl tissues infected by Pythium ultimum was examined by light and electron microscopy, and by energy dispersive X-ray analysis (EDX). Plants were grown in 0 or 1·7 m Si-amended nutrient solutions, and root and hypocotyl samples were collected at different times after inoculation with P. ultimum. By 48 h after infection, striking differences in the expression of defence reactions were observed between Si-amended and Si-free cucumber plants. Treatment of plants with Si markedly stimulated the accumulation of an electron-dense, phenolic-like material in infected host tissues, and significantly increased the percentage of cells filled with this material. Fungal hyphae colonizing occluded host cells were seriously damaged, and were often reduced to empty hyphal shells. Additionally, Si-treated cucumber plants responded to P. ultimum infection by forming electron-dense layers along primary and secondary cell walls, as well as over pit membranes of xylem vessels. EDX analysis failed to reveal the presence of silica deposits in P. ultimum-infected plants grown in Si-supplemented media. Our results suggest that a relationship exists between Si treatment, resistance to P. ultimum attack, and expression of plant defence mechanisms.     

Efficacy of UV‐C radiation to reduce seedborne anthracnose (Colletotrichum acutatum) from Andean lupin (Lupinus mutabilis)

The potential of UV‐C radiation of Andean lupin (Lupinus mutabilis) seeds to eradicate seedborne infections of anthracnose caused by Colletotrichum acutatum was investigated. UV‐C doses from 0 to 691.2 kJ m^?2 (resulting from 0 to 96 h of exposure time) on disease incidence reduction and germination on artificially and naturally infected seed were evaluated. The degree of incidence reduction and seed germination was dependent on the dose of UV‐C. The UV‐C doses of 86.4 kJ m^?2 and higher reduced incidence from 6% to 7% to undetectable levels, but these UV‐C doses also reduced seed germination. UV‐C can deleteriously affect physiological processes and overall growth. To assess its impact, L. mutabilis seeds irradiated with UV‐C doses of 57.6 and 86.4 kJ m^?2 were grown. Seedlings grown from noninfected seed and UV‐C treated seed showed an increased concentration of chlorophyll and protein contents, as well as an increase in the activation of defence enzymes peroxidase and catalase, in comparison with plants grown from infected seed. UV‐C doses resulted in seed emergence and seedling dry weight rates that were similar to the noninfected control or better than the fungicide control. Moreover, 57.6 kJ m^?2 reduced transmission of the pathogen from seed to the plantlets by 80%, while 86.4 kJ m^?2 apparently eradicated the pathogen, under greenhouse conditions. The use of UV‐C, first reported here, is advantageous for controlling anthracnose in lupin.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.